U.S. patent application number 16/950486 was filed with the patent office on 2021-10-14 for inhibitors of kidney-type glutaminase, gls-1.
The applicant listed for this patent is CORNELL UNIVERSITY, ITHACA COLLEGE. Invention is credited to Kristin CERIONE, Richard CERIONE, Clint STALNECKER, Scott ULRICH.
Application Number | 20210317115 16/950486 |
Document ID | / |
Family ID | 1000005666124 |
Filed Date | 2021-10-14 |
United States Patent
Application |
20210317115 |
Kind Code |
A1 |
CERIONE; Richard ; et
al. |
October 14, 2021 |
INHIBITORS OF KIDNEY-TYPE GLUTAMINASE, GLS-1
Abstract
The present invention relates generally to glutaminase
inhibitors of Formula I, Formula II, or Formula III, as well as
pharmaceutical compounds containing them and methods of their
use.
Inventors: |
CERIONE; Richard; (Ithaca,
NY) ; CERIONE; Kristin; (Ithaca, NY) ;
STALNECKER; Clint; (Ithaca, NY) ; ULRICH; Scott;
(Brooktondale, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CORNELL UNIVERSITY
ITHACA COLLEGE |
Ithaca
Ithaca |
NY
NY |
US
US |
|
|
Family ID: |
1000005666124 |
Appl. No.: |
16/950486 |
Filed: |
November 17, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16735479 |
Jan 6, 2020 |
10889585 |
|
|
16950486 |
|
|
|
|
15533198 |
Jun 5, 2017 |
10526322 |
|
|
PCT/US2015/064152 |
Dec 5, 2015 |
|
|
|
16735479 |
|
|
|
|
62088370 |
Dec 5, 2014 |
|
|
|
62102163 |
Jan 12, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07C 211/49 20130101;
C12Q 1/34 20130101; C07D 221/04 20130101; C07C 211/59 20130101;
C07D 277/82 20130101; C07C 311/21 20130101; C07C 211/58 20130101;
C07C 311/37 20130101; C07D 233/61 20130101; C12N 9/96 20130101;
C07C 217/90 20130101; C07D 221/06 20130101; C07D 401/10 20130101;
C07C 237/40 20130101; C12Y 305/01002 20130101; C07D 295/135
20130101; C07D 221/18 20130101; C07C 211/52 20130101; C12N 9/80
20130101; C07D 207/16 20130101; C07D 221/12 20130101; G01N 2333/98
20130101; C07D 471/04 20130101 |
International
Class: |
C07D 471/04 20060101
C07D471/04; C07D 221/18 20060101 C07D221/18; C07D 207/16 20060101
C07D207/16; C07D 277/82 20060101 C07D277/82; C07D 295/135 20060101
C07D295/135; C07C 211/58 20060101 C07C211/58; C07C 217/90 20060101
C07C217/90; C07C 237/40 20060101 C07C237/40; C07D 221/06 20060101
C07D221/06; C07D 401/10 20060101 C07D401/10; C07C 311/37 20060101
C07C311/37; C07C 211/49 20060101 C07C211/49; C07C 211/52 20060101
C07C211/52; C07D 233/61 20060101 C07D233/61; C07C 211/59 20060101
C07C211/59; C07C 311/21 20060101 C07C311/21; C07D 221/04 20060101
C07D221/04; C07D 221/12 20060101 C07D221/12; C12N 9/80 20060101
C12N009/80; C12N 9/96 20060101 C12N009/96; C12Q 1/34 20060101
C12Q001/34 |
Claims
1. A compound, or a pharmaceutically acceptable salt, ester, enol
ether, enol ester, solvate, hydrate, or prodrug thereof, wherein
the compound is selected from the group consisting of: (i) a
compound of Formula I: ##STR00022## wherein: R is selected from the
group consisting of monocyclic or bicyclic aryl, monocyclic or
bicyclic heteroaryl, and monocyclic or bicyclic heterocyclyl,
wherein each monocyclic or bicyclic aryl, monocyclic or bicyclic
heteroaryl, and monocyclic or bicyclic heterocyclyl can be
optionally substituted from 1 to 4 times with substituents
independently selected at each occurrence thereof from the group
consisting of H, halogen, C.sub.1-6 alkyl, aryl, --OR.sup.8,
--CF.sub.3, and --CHF.sub.2; R.sup.1 and R.sup.2 are each
independently selected from the group consisting of H, halogen, and
C.sub.1-6 alkyl; or R.sup.1 and R.sup.2 are combined to form
.dbd.O; R.sup.3--R.sup.7 are each independently selected from the
group consisting of H, halogen, --NO.sub.2, --NR.sup.8R.sup.9,
--SO.sub.2NR.sup.8R.sup.9, --N.sub.3, --C(O)R.sup.8, aryl,
heteroaryl, heterocyclyl, ##STR00023## and R.sup.8 and R.sup.9 are
each independently selected from the group consisting of H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, and aryl; or
R.sup.8 and R.sup.9 are combined with the nitrogen to which they
are attached to form a heterocyclyl, wherein the heterocyclyl can
be optionally substituted with --COOH or --COOMe; (ii) a compound
of Formula II: ##STR00024## wherein: the dotted circle identifies
an active moiety; X is independently --CR.sub.14a-- or --N;
R.sub.1a is independently H, --OH, --OR.sub.14a, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl,
R.sub.14aC(O)--, R.sub.14aOC(O)--, R.sub.14aS(O)--, or
R.sub.14aS(O).sub.2--; R.sub.2a, R.sub.3a, R.sub.4a, R.sub.5a, and
R.sub.6a are each independently a photoreactive moiety, H, halogen,
--NO.sub.2, --OH, --OR.sub.14a, --SR.sub.14a, --NH.sub.2,
--NHR.sub.14a, --NR.sub.14aR.sub.15a, R.sub.14aC(O)--,
R.sub.14aOC(O), R.sub.14aC(O)O--, C.sub.1-C.sub.6 alkyl, C.sub.2-6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono
or polycyclic aryl, or mono or polycyclic heteroaryl with each
cyclic unit containing from 1 to 5 heteroatoms selected from the
group consisting of nitrogen, sulfur, and oxygen, wherein the
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl,
mono or polycyclic aryl, and mono or polycyclic heteroaryl are
optionally substituted with a photoreactive moiety; or R.sub.2a and
R.sub.3a, R.sub.3a and R.sub.4a, R.sub.4a and R.sub.5a, or R.sub.5a
and R.sub.6a are combined to form a heterocyclic ring optionally
substituted with a photoreactive moiety; R.sub.7a, R.sub.8a,
R.sub.9a, and R.sub.10a are each independently a photoreactive
moiety, H, --OH, --NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono
or polycyclic aryl, or mono or polycyclic heteroaryl with each
cyclic unit containing from 1 to 5 heteroatoms selected from the
group consisting of nitrogen, sulfur, and oxygen, wherein the aryl,
heteroaryl, and aryl C.sub.1-C.sub.6 alkyl are optionally
substituted from 1 to 3 times with substituents selected from the
group consisting of, halogen, --OH, --NH.sub.2, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkoxy, --SH, and
C.sub.1-C.sub.6 thioalkyl, and wherein the alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl,
and mono or polycyclic heteroaryl are optionally substituted with a
photoreactive moiety; and R.sub.11a, R.sub.12a, R.sub.13a,
R.sub.14a, R.sub.15a, R.sub.16a, and R.sub.17a are each
independently a photoreactive moiety, H, halogen, --OH, --NO.sub.2,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7
cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic
aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkylalkyl, arylalkyl, and mono or polycyclic aryl are
optionally substituted with a photoreactive moiety and each one of
R.sub.11a-R.sub.17a is optionally substituted with --NH.sub.2,
--OH, halogen, --COOH, --NO.sub.2, and --CN; and wherein the
compound comprises at least one photoreactive moiety; and (iii) a
compound of Formula III: ##STR00025## wherein: R is selected from
the group consisting of monocyclic or bicyclic aryl, monocyclic or
bicyclic heteroaryl, and monocyclic or bicyclic heterocyclyl,
wherein each monocyclic or bicyclic aryl, monocyclic or bicyclic
heteroaryl, and monocyclic or bicyclic heterocyclyl can be
optionally substituted from 1 to 4 times with substituents
independently selected at each occurrence thereof from the group
consisting of H, halogen, C.sub.1-6 alkyl, aryl, --OR.sup.8,
--CF.sub.3, and --CHF.sub.2; R.sup.1 and R.sup.2 are each
independently selected from the group consisting of a photoreactive
moiety, H, halogen, and C.sub.1-6 alkyl optionally substituted with
a photoreactive moiety; or R.sup.1 and R.sup.2 are combined to form
.dbd.O; R.sup.3--R.sup.7 are each independently selected from the
group consisting of a photoreactive moiety, H, halogen, --NO.sub.2,
--NR.sup.8R.sup.9, --SO.sub.2NR.sup.8R.sup.9, --N.sub.3,
--C(O)R.sup.8, aryl, heteroaryl, and heterocyclyl, wherein the aryl
and heteroaryl are optionally substituted with a photoreactive
moiety; and R.sup.8 and R.sup.9 are each independently selected
from the group consisting of a photoreactive moiety, H, C.sub.1-6
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, and aryl, wherein the
alkyl, alkenyl, alkynyl, and aryl are optionally substituted with a
photoreactive moiety; or R.sup.8 and R.sup.9 are combined with the
nitrogen to which they are attached to form a heterocyclyl, wherein
the heterocyclyl can be optionally substituted with a photoreactive
moiety, --COOH, or --COOMe; and wherein the compound comprises at
least one photoreactive moiety; and wherein the compound is
optionally modified to include a tag and/or an attachment to a
solid surface.
2. The compound of claim 1, wherein the compound is a compound of
Formula I.
3-4. (canceled)
5. The compound according to claim 1, wherein the compound is a
compound of Formula II or Formula III.
6. The compound according to claim 5, wherein the photoreactive
moiety is selected from the group consisting of aryl azides,
diazirines, and benzophenone.
7. The compound according to claim 6, wherein the photoreactive
moiety is selected from the group consisting of
--N.dbd.N.sup.+.dbd.N.sup.-; ##STR00026##
8. The compound according to claim 5, wherein the compound is a
compound of Formula II.
9. (canceled)
10. The compound according to claim 5, wherein the compound is a
compound of Formula III.
11. (canceled)
12. A pharmaceutical composition comprising: a compound of claim 1,
or a pharmaceutically acceptable salt, ester, enol ether, enol
ester, solvate, hydrate, or prodrug thereof.
13. The pharmaceutical composition of claim 12 further comprising:
a pharmaceutically acceptable carrier.
14. A method of treating a subject with a condition mediated by
production of glutamate from glutamine by glutaminase GLS1, said
method comprising: selecting a subject with a condition mediated by
production of glutamate from glutamine by glutaminase GLS1 and
administering to said selected subject an inhibitor of glutaminase
GLS1 activity under conditions effective to treat the condition
mediated by production of glutamate from glutamine, wherein the
inhibitor is a compound of claim 1, or a pharmaceutically
acceptable salt, ester, enol ether, enol ester, solvate, hydrate,
or prodrug thereof.
15. The method of claim 14, wherein the condition is a cancer that
exhibits active glutaminase GLS1 activity.
16. The method of claim 15, wherein the cancer is selected from the
group consisting of breast cancer, brain cancer, lung cancer,
ovarian cancer, pancreatic cancer, colon cancer, and multiple
myeloma.
17-21. (canceled)
22. A method of reducing the production of glutamate from glutamine
by glutaminase GLS1 in a sample, said method comprising: inhibiting
glutaminase GLS1 activity in the sample by a method comprising:
providing a compound and contacting glutaminase GLS1 in the sample
with the compound to reduce the production of glutamate from
glutamine in the sample, wherein the compound is a compound of
claim 1, or a pharmaceutically acceptable salt, ester, enol ether,
enol ester, solvate, hydrate, or prodrug thereof.
23. A method of detecting glutaminase GLS1 protein in a sample,
said method comprising: providing a sample potentially containing
glutaminase GLS1 protein; contacting the sample with a compound
comprising a photoreactive moiety; exposing the compound to a light
source under conditions effective to form a conjugate between the
compound and glutaminase GLS1 protein, if present in the sample,
through covalent modification of the photoreactive moiety; and
detecting whether any compound-glutaminase GLS1 protein conjugates
are formed, wherein formation of a compound-glutaminase GLS1
protein conjugate indicates the presence of glutaminase GLS1
protein in the sample; wherein the compound is a compound of claim
5, or a pharmaceutically acceptable salt, ester, enol ether, enol
ester, solvate, hydrate, or prodrug thereof.
24. A method of producing a glutaminase inhibitor-glutaminase GLS1
protein conjugate in a sample; providing a sample containing one of
(i) glutaminase GLS1 protein and (ii) a compound comprising a
photoreactive moiety; contacting the sample with the other of (i)
glutaminase GLS1 protein and (ii) a compound comprising a
photoreactive moiety; and exposing the compound to a light source
under conditions effective to form a conjugate between the compound
and glutaminase GLS1 protein through covalent modification of the
photoreactive moiety; wherein the compound is a compound of claim
5, or a pharmaceutically acceptable salt, ester, enol ether, enol
ester, solvate, hydrate, or prodrug thereof.
25. The method according to claim 24 further comprising: (i)
detecting the compound, the conjugate, and/or the glutaminase GLS1
protein; (ii) quantitating the amount of compound, the conjugate,
and/or the glutaminase GLS1 protein present in the sample; (iii)
isolating the compound, the conjugate, and/or the glutaminase GLS1
protein from the sample; (iv) purifying the compound, the
conjugate, and/or the glutaminase GLS1 protein; or (v) any
combination thereof.
26-39. (canceled)
40. The method according to claim 14, wherein the glutaminase GLS1
is glutaminase C ("GAC").
41. The method according to claim 14, wherein the glutaminase GLS1
is brain glutaminase ("KGA").
42. The compound according to claim 1, wherein the compound is
modified to include a tag and/or an attachment to a solid
surface.
43. A pharmaceutically acceptable salt, ester, enol ether, enol
ester, solvate, hydrate, or prodrug of a compound according to
claim 1.
Description
[0001] This application is continuation of U.S. patent application
Ser. No. 16/735,479, filed Jan. 6, 2020, which is a continuation of
U.S. patent application Ser. No. 15/533,198, filed Jun. 5, 2017,
which issued as U.S. Pat. No. 10,526,322 and is a national stage
application under 35 U.S.C. .sctn. 371 of International Patent
Application No. PCT/US2015/064152, filed Dec. 5, 2015, which claims
the benefit of U.S. Provisional Patent Application Ser. No.
62/088,370, filed Dec. 5, 2014, and U.S. Provisional Patent
Application Ser. No. 62/102,163, filed Jan. 12, 2015, each of which
is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to inhibitors of
glutaminase.
BACKGROUND OF THE INVENTION
[0003] Tumor cells have an absolute requirement for glutamine as a
growth substrate. Glutamine is required as a precursor for both DNA
synthesis and protein synthesis, and may also be used as a
respiratory substrate. In experiments where glutamine metabolism in
tumor cells has been specifically compared with that in
non-transformed cells of the same origin, glutamine metabolism in
the tumor cells has been found to be considerably faster. This is
true for human hepatocytes and hepatoma cells (Souba, W.,
"Glutamine and Cancer," Ann. Surg. 218:715-28 (1993)) and also for
glutamine oxidation in rat kidney fibroblasts and rat fibrosarcoma
cells (Fischer et al., "Adaptive Alterations in Cellular Metabolism
and Malignant Transformation," Ann. Surg. 227:627-34 (1998)).
[0004] The first reaction in glutamine metabolism is hydrolysis of
glutamine to glutamate via the mitochondrial enzyme
phosphate-dependent glutaminase. Two major isoforms of this enzyme
have been characterized. These are known as the kidney form
(K-type) which was first cloned from rat kidney (Shapiro et al.,
"Isolation, Characterisation, and In vitro Expression of a cDNA
That Encodes the Kidney Isoenzyme of the Mitochondrial
Glutaminase," J. Biol. Chem. 266:18792-96 (1991)) and is expressed
in many mammalian tissues, and the liver form (L-type) (Chung-Bok
et al., "Rat Hepatic Glutaminase, Identification of the Full Coding
Sequence and Characterisation of a Functional Promoter," Biochem.
J. 324:193-200 (1997)) which was originally identified in
post-natal liver. These two enzymes have different kinetic
properties. A splice variant of the K-type, Glutaminase C (GAC),
has also been identified and both are commonly referred to as
GLS1.
[0005] Although the cDNAs encoding the two isoforms have regions of
high sequence similarity, they also differ significantly elsewhere
and the enzyme isoforms are the products of different genes (for a
review see (Curthoys et al., "Regulation of Glutaminase Activity
and Glutamine Metabolism," Annu. Rev. Nutr. 16:133-59 (1995)).
Glutamine metabolism is essential for tumor cell growth but there
are few studies at present on glutaminase expression in tumor
cells. In mouse Ehrlich ascites cells (Quesada et al.,
"Purification of Phosphate-Dependent Glutaminase from Isolated
Mitochondria of Ehrlich Ascites-Tumor Cells," Biochem. J.
255:1031-35 (1988)) and rat fibrosarcoma cells (Fischer et al.,
"Adaptive Alterations in Cellular Metabolism and Malignant
Transformation," Ann. Surg. 227:627-34 (1998)), an enzyme with the
kinetic properties of the K-type glutaminase is expressed. Rat and
human hepatocytes express the L-type glutaminase, but this is not
expressed in hepatoma cell lines, which express the K-type instead
(Souba, W. W., "Glutamine and Cancer," Ann. Surg. 218:715-28
(1993)). Inhibition of K-type glutaminase expression by anti-sense
mRNA in Ehrlich ascites cells has been shown to decrease the growth
and tumorigenicity of these cells (Lobo et al., "Inhibition of
Glutaminase Expression by Antisense mRNA Decreases Growth and
Tumorigenicity of Tumor Cells," Biochem. J. 348:257-61 (2000)).
[0006] Since it is well-known that tumorigenesis is linked to
glutamine metabolism, the present invention can have an important
impact in cancer therapeutics.
[0007] The present invention is directed to overcoming these and
other deficiencies in the art.
SUMMARY OF THE INVENTION
[0008] A first aspect of the present invention relates to a
compound, or a pharmaceutically acceptable salt, ester, enol ether,
enol ester, solvate, hydrate, or prodrug thereof, wherein the
compound is a compound of Formula I:
##STR00001##
wherein: [0009] R is selected from the group consisting of
monocyclic or bicyclic aryl, monocyclic or bicyclic heteroaryl, and
monocyclic or bicyclic heterocyclyl, wherein each monocyclic or
bicyclic aryl, monocyclic or bicyclic heteroaryl, and monocyclic or
bicyclic heterocyclyl can be optionally substituted from 1 to 4
times with substituents independently selected at each occurrence
thereof from the group consisting of H, halogen, C.sub.1-6 alkyl,
aryl, --OR.sup.8, --CF.sub.3, and --CHF.sub.2; [0010] R.sup.1 and
R.sup.2 are each independently selected from the group consisting
of H, halogen, and C.sub.1-6 alkyl; or R.sup.1 and R.sup.2 are
combined to form .dbd.O; [0011] R.sup.3-R.sup.7 are each
independently selected from the group consisting of H, halogen,
--NO.sub.2, --NR.sup.8R.sup.9, --SO.sub.2NR.sup.8R.sup.9,
--N.sub.3, --C(O)R.sup.8, aryl, heteroaryl, heterocyclyl,
##STR00002##
[0011] and [0012] R.sup.8 and R.sup.9 are each independently
selected from the group consisting of H, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and aryl; or R.sup.8 and R.sup.9 are
combined with the nitrogen to which they are attached to form a
heterocyclyl, wherein the heterocyclyl can be optionally
substituted with --COOH or --COOMe; and wherein the compound is
optionally modified to include a tag and/or an attachment to a
solid surface.
[0013] A second aspect of the present invention relates to a
compound, or a pharmaceutically acceptable salt, ester, enol ether,
enol ester, solvate, hydrate, or prodrug thereof, wherein the
compound is a compound of Formula II:
##STR00003## [0014] wherein: [0015] the dotted circle identifies an
active moiety; [0016] X is independently --CR.sub.14a-- or --N;
[0017] R.sub.1a is independently H, --OH, --OR.sub.14a,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, R.sub.14aC(O)--, R.sub.14aOC(O)--, R.sub.14aS(O)--, or
R.sub.14aS(O).sub.2.sup.-, [0018] R.sub.2a, R.sub.3a, R.sub.4a,
R.sub.5a, and R.sub.6a are each independently a photoreactive
moiety, H, halogen, --NO.sub.2, --OH, --OR.sub.14a, --SR.sub.14a,
--NH.sub.2, --NHR.sub.14a, --NR.sub.14aR.sub.15a, R.sub.14aC(O)--,
R.sub.14aOC(O)--, R.sub.14aC(O)O--, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6
alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl
with each cyclic unit containing from 1 to 5 heteroatoms selected
from the group consisting of nitrogen, sulfur, and oxygen, wherein
the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl,
arylalkyl, mono or polycyclic aryl, and mono or polycyclic
heteroaryl are optionally substituted with a photoreactive moiety;
or R.sub.2a and R.sub.3a, R.sub.3a and R.sub.4a, R.sub.4a and
R.sub.5a, or R.sub.5a and R.sub.6a are combined to form a
heterocyclic ring optionally substituted with a photoreactive
moiety; [0019] R.sub.7a, R.sub.8a, R.sub.9a, and R.sub.10a are each
independently a photoreactive moiety, H, --OH, --NH.sub.2,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7
cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic
aryl, or mono or polycyclic heteroaryl with each cyclic unit
containing from 1 to 5 heteroatoms selected from the group
consisting of nitrogen, sulfur, and oxygen, wherein the aryl,
heteroaryl, and aryl C.sub.1-C.sub.6 alkyl are optionally
substituted from 1 to 3 times with substituents selected from the
group consisting of, halogen, --OH, --NH.sub.2, C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkoxy, --SH, and
C.sub.1-C.sub.6 thioalkyl, and wherein the alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkylalkyl, arylalkyl, mono or polycyclic aryl,
and mono or polycyclic heteroaryl are optionally substituted with a
photoreactive moiety; and [0020] R.sub.11a, R.sub.12a, R.sub.13a,
R.sub.14a, R.sub.15a, R.sub.16a, and R.sub.17a are each
independently a photoreactive moiety, H, halogen, --OH, --NO.sub.2,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7
cycloalkylalkyl, aryl C.sub.1-C.sub.6 alkyl, mono or polycyclic
aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkylalkyl, arylalkyl, and mono or polycyclic aryl are
optionally substituted with a photoreactive moiety and each one of
R.sub.11a-R.sub.17a is optionally substituted with --NH.sub.2,
--OH, halogen, --COOH, --NO.sub.2, and --CN; [0021] wherein the
compound comprises at least one photoreactive moiety; and [0022]
wherein the compound is optionally modified to include a tag and/or
an attachment to a solid surface.
[0023] A third aspect of the present invention relates to a
compound, or a pharmaceutically acceptable salt, ester, enol ether,
enol ester, solvate, hydrate, or prodrug thereof, wherein the
compound is a compound of Formula III:
##STR00004## [0024] wherein: [0025] R is selected from the group
consisting of monocyclic or bicyclic aryl, monocyclic or bicyclic
heteroaryl, and monocyclic or bicyclic heterocyclyl, wherein each
monocyclic or bicyclic aryl, monocyclic or bicyclic heteroaryl, and
monocyclic or bicyclic heterocyclyl can be optionally substituted
from 1 to 4 times with substituents independently selected at each
occurrence thereof from the group consisting of H, halogen,
C.sub.1-6 alkyl, aryl, --OR.sup.8, --CF.sub.3, and --CHF.sub.2;
[0026] R.sup.1 and R.sup.2 are each independently selected from the
group consisting of a photoreactive moiety, H, halogen, and
C.sub.1-6 alkyl optionally substituted with a photoreactive moiety;
or R.sup.1 and R.sup.2 are combined to form .dbd.O; [0027]
R.sup.3-R.sup.7 are each independently selected from the group
consisting of a photoreactive moiety, H, halogen, --NO.sub.2,
--NR.sup.8R.sup.9, --SO.sub.2NR.sup.8R.sup.9, --N.sub.3,
--C(O)R.sup.8, aryl, heteroaryl, and heterocyclyl, wherein the aryl
and heteroaryl are optionally substituted with a photoreactive
moiety; and [0028] R.sup.8 and R.sup.9 are each independently
selected from the group consisting of a photoreactive moiety, H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, and aryl,
wherein the alkyl, alkenyl, alkynyl, and aryl are optionally
substituted with a photoreactive moiety; or R.sup.8 and R.sup.9 are
combined with the nitrogen to which they are attached to form a
heterocyclyl, wherein the heterocyclyl can be optionally
substituted with a photoreactive moiety, --COOH, or --COOMe; [0029]
wherein the compound comprises at least one photoreactive moiety;
and [0030] wherein the compound is optionally modified to include a
tag and/or an attachment to a solid surface.
[0031] A fourth aspect of the present invention relates to a
pharmaceutical composition comprising a compound of Formula I,
Formula II, or Formula III, or a pharmaceutically acceptable salt,
ester, enol ether, enol ester, solvate, hydrate, or prodrug
thereof.
[0032] A fifth aspect of the present invention relates to a method
of reducing the production of glutamate from glutamine in a sample.
This method involves inhibiting glutaminase GLS1 activity in the
sample by providing a compound and contacting glutaminase GLS1 in
the sample with the compound to reduce the production of glutamate
from glutamine in the sample, wherein the compound is a compound of
Formula I, Formula II, or Formula III, or a pharmaceutically
acceptable salt, ester, enol ether, enol ester, solvate, hydrate,
or prodrug thereof.
[0033] A sixth aspect of the present invention relates to a method
of treating a subject with a condition mediated by production of
glutamate from glutamine by glutaminase GLS1. The method includes
selecting a subject with a condition mediated by production of
glutamate from glutamine by glutaminase GLS1 and administering to
the selected subject an inhibitor of glutaminase GLS1 activity
under conditions effective to treat the condition mediated by
production of glutamate from glutamine, wherein the inhibitor is a
compound of Formula I, Formula II, or Formula III, or a
pharmaceutically acceptable salt, ester, enol ether, enol ester,
solvate, hydrate, or prodrug thereof.
[0034] A seventh aspect of the present invention relates to methods
that involve the formation of a conjugate between a compound and
glutaminase GLS1 protein. One embodiment of this aspect of the
present invention relates to a method of detecting glutaminase GLS1
protein in a sample. This embodiment involves providing a sample
potentially containing glutaminase GLS1 protein; contacting the
sample with a compound comprising a photoreactive moiety; exposing
the compound to a light source under conditions effective to form a
conjugate between the compound and glutaminase GLS1 protein, if
present in the sample, through covalent modification of the
photoreactive moiety; and detecting whether any
compound-glutaminase GLS1 protein conjugates are formed, wherein
formation of a compound-glutaminase GLS1 protein conjugate
indicates the presence of glutaminase GLS1 protein in the sample;
and wherein the compound is a compound of Formula II or III, or a
pharmaceutically acceptable salt, ester, enol ether, enol ester,
solvate, hydrate, or prodrug thereof. Another embodiment of this
aspect of the present invention relates to a method of producing a
glutaminase inhibitor-glutaminase GLS1 protein conjugate in a
sample. This embodiment involves providing a sample containing one
of (i) glutaminase GLS1 protein and (ii) a compound comprising a
photoreactive moiety; contacting the sample with the other of (i)
glutaminase GLS1 protein and (ii) a compound comprising a
photoreactive moiety; and exposing the compound to a light source
under conditions effective to form a conjugate between the compound
and glutaminase GLS1 protein through covalent modification of the
photoreactive moiety; wherein the compound is a compound of Formula
II or III, or a pharmaceutically acceptable salt, ester, enol
ether, enol ester, solvate, hydrate, or prodrug thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIGS. 1A-D show that Dbl-induced transformation and
increased glutaminolysis are inhibited by 968. FIG. 1A shows
fluorescent staining before (+Dox) and after (-Dox) a 24-hour
induction of Dbl-inducible MEFs with anti-actin (top) and anti-HA
(bottom) antibodies. FIG. 1B shows that expression of Dbl confers
the ability of MEFs to form foci, which is blocked by treatment
with 10 .mu.M 968. FIG. 1C is a diagram showing .sup.13C enrichment
from [U-.sup.13C]-glutamine into TCA cycle intermediates, where GAC
activation downstream from Dbl is highlighted. .sup.13C-carbons are
shown as dark-filled circles and .sup.12C-carbons as light-filled
circles. FIG. 1D shows glutamine-derived metabolites (glutamate
M+5, fumarate M+4, malate M+4, citrate M+4) normalized to .sup.13C
enrichment observed for MEFs not expressing Dbl. Comparisons were
made between treatment with 968, its less potent analog 27, and
untreated cells. Bars represent the mean (.+-.SD) of triplicate
determinations. P-values were determined by the Students t-test (*
p<0.05, ** p<0.005).
[0036] FIGS. 2A-H are histograms (FIGS. 2A and 2C-H) and a .sup.13C
enrichment diagram (FIG. 2B) relating to the measured isotopologues
of TCA cycle intermediates derived from [U-.sup.13C]glutamine or
[U-.sup.13C]glucose in non-induced and induced Dbl-MEFs with 968
and 27 treatment. FIG. 2A shows that 968 treatment (8 .mu.M) causes
a modest decrease in glutamate pool sizes in non-induced and
induced cells while having no effect on the pool sizes of fumarate,
malate, and citrate. Metabolites were quantified by normalizing the
integrated peaks for all mass isotopologues with respect to the
internal standard (50 nmol of 2-oxobutyrate) and protein content.
FIG. 2B shows the incorporation of glucose- and glutamine-derived
carbon into TCA cycle intermediates. Glutamine-derived carbons are
shown as dark-filled circles and glucose derived carbons as
light-filled circles. FIGS. 2C-F show isolated and quantified TCA
cycle isotopologues following a 1 hour incubation of Dbl-MEFs with
[U-.sup.13C]-glutamine in both the induced (-DOX) and non-induced
states (+DOX), with overnight treatment of 8 .mu.M 968, 27 or DMSO
control to illustrate effects on glutamine metabolism (+)/(-) DOX
as well as with drug treatments. [U-.sup.13C]glutamine enrichment
is noted in the M+5 isotopologue of glutamate (FIG. 2C), M+4 of
fumarate (FIG. 2D), M+4 of malate (FIG. 2E), and M+4 of citrate
(FIG. 2F), where an inhibition of glutamine metabolism by 968 was
observed in both induced and non-induced cells as read out by
.sup.13C enrichment. FIGS. 2G-H show isolated and quantified
citrate isotopologues following incubation of [U-.sup.13C]glucose
in Dbl-MEFs in both the induced and non-induced states to
illustrate the effects of Dbl-induction on glucose fueled
anaplerosis both over time and with drug treatments. FIG. 2G shows
the kinetics of [U-.sup.13C]glucose enrichment of the TCA cycle
intermediate citrate in the induced and non-induced states as read
out by the increase of M+2 over time. FIG. 2H shows 968 inhibition
of [U-.sup.13C]glucose enrichment in the M+2 isotopologue of
citrate following an 8 hour incubation of Dbl-MEFs with
[U-.sup.13C]glucose in the induced and noninduced states, with
overnight treatment of 8 .mu.M 968 and 27; this is thought to be
due to the inhibition of glutamine derived TCA cycle intermediates
being incorporated into the primary substrate of glucose
incorporation into the TCA cycle, oxaloacetate (OAA).
[0037] FIGS. 3A-F are a schematic diagram (FIG. 3A) and graphs
(FIGS. 3B-F) that relate to a real-time fluorescence assay for
detecting GAC tetramer formation. FIG. 3A is a schematic depiction
of the FRET assay. FIG. 3B shows that 25 nM 488-GAC (donor)
fluorescence is quenched upon addition of QSY9-GAC (acceptor) in a
dose-dependent manner and reversed with the addition of a 10-fold
excess of unlabeled GAC. FIG. 3C shows FRET resulting from the
titration of 25 nM 488-GAC with increasing amounts of QSY9-GAC
(open circles) overlaid with concentration-dependent in vitro
activation of GAC (closed circles). FRET data was fit to a
quadratic binding isotherm. Points represent the mean.+-.SD of
three independent experiments. FIG. 3D shows increasing amounts of
BPTES added to 25 nM 488-GAC and 25 nM QSY9-GAC to examine the
effects of the inhibitor on GAC tetramer formation. A 10-fold
excess of unlabeled GAC was added to attempt to reverse tetramer
formation. FIG. 3E shows that 968 induces a dose-dependent
quenching of 488-GAC fluorescence that is distinct from the
quenching induced by the addition of QSY9-GAC. FIG. 3F shows
fluorescence quenching upon addition of different concentrations of
968 to 10 nM 488-GAC in the absence of QSY9-GAC.
[0038] FIG. 4 is a graph showing quenching of 20 nM 488-labeled GAC
fluorescence by 968 (.circle-solid.) and inhibition of 20 nM
unlabeled WT GAC as measured by NADH fluorescence emission
(.smallcircle.), as described for FIG. 5A, where unlabeled WT GAC
(20 nM) was assayed in place of 488-labeled GAC.
[0039] FIGS. 5A-E relate to the development of real-time 968
binding and inhibition assays. FIG. 5A is a schematic model of
real-time 968 binding and inhibition assays. Monitoring 488-GAC
fluorescence quenching serves as a read-out for 968 binding, and
enzymatic activity is monitored through the generation of NADH
fluorescence upon addition of 20 mM glutamine and 50 mM phosphate
to an assay incubation containing labeled GAC together with 10
units of glutamate dehydrogenase (GDH) and 2 mM NAD+. FIG. 5B is a
graph of fluorescence of 10 nM 488-GAC (520 nm emission, "a"
curves) monitored upon addition of 20 .mu.M 968 (-), 10 .mu.M BPTES
(.circle-solid..circle-solid..circle-solid.), or DMSO (---) at the
indicated time. Simultaneously, NADH fluorescence (460 nm emission,
"b" curves) was monitored following the addition of 20 mM glutamine
and 50 mM phosphate at 120 seconds. FIG. 5C is a graph of real-time
968 binding and inhibition assays adapted to a 96-well plate format
and shows overlapping inhibition and fluorescence quenching
profiles for 10 nM 488-GAC and 10 nM wild-type (WT) unlabeled GAC.
Data points are the average .+-.SD of three independent
experiments. The solid line shows the semi-log plot of the binding
isotherm with K.sub.D=3 .mu.M. FIG. 5D shows the structures of 968
and 968-like analogues used in real-time binding and inhibition
assays. FIG. 5E shows plotted IC.sub.50 (.+-.SD) values from
inhibition data and measured K.sub.D (.+-.SD) values from
fluorescence quenching data for a representative group of 968
analogues (depicted in FIG. 5D). The compounds a-i correspond to
the letter designations shown in FIG. 5D. Values obtained from
inhibition data and quenching data were fit to a ligand binding
equation for a biomolecular interaction. The line represents a
linear regression fit with the following values: R.sup.2=0.92,
slope=1.10.
[0040] FIGS. 6A-D show that binding of 968 is not affected by
pretreatment of GAC with the allosteric activator inorganic
phosphate whereas its inhibitory potency is markedly reduced. FIG.
6A is a cartoon model of the FRET and 968-binding assays. FIG. 6B
is an emission spectra that shows that relative fluorescence
emission of 25 nM 488-GAC, in the presence (broken-dotted line) or
absence (solid and broken lines) of 100 mM P.sub.1, is quenched
upon addition of 25 nM QSY9-GAC. 488-GAC fluorescence emission was
further quenched upon addition of 10 .mu.M 968, compared to the
DMSO control, as a result of 968 binding. As shown in FIGS. 6C-D,
10 nM 488-GAC was assayed for 968 binding (quantified in FIG. 6C)
and inhibition (quantified in FIG. 6D), using the assays depicted
in FIG. 6A, where 50 mM P.sub.i was added either prior to, or
after, 968 addition. Data points represent the average (.+-.SD) of
3 independent experiments, and were fit to a ligand binding
equation for a biomolecular interaction.
[0041] FIGS. 7A-G relate to the examination of 968 binding to
monomeric and dimeric GAC mutants. FIG. 7A shows the crystal
structure of the GAC tetramer (human isoform) in complex with both
BPTES and glutamate (PDB 3U09), with the proposed 968-binding
pocket indicated by the arrow pointing toward the C-terminal
monomer-monomer interface. Insets highlight critical
monomer-monomer (top) and dimer-dimer (bottom) contacts, with the
corresponding human and mouse GAC isoform residue numbering. FIG.
7B shows multi-angle light scattering profiles of WT GAC (a),
D391K-GAC (b), and K316E-D391K-R459E-GAC (c), 250 .mu.g (each),
where the solid line represents the elution of each species by
monitoring refractive index (R.I.), and the broken line designates
the calculated molecular weight for the species eluted at that
time. Reference lines for the molecular weights of the monomeric,
dimeric, and tetrameric forms of the enzyme are included at 58 kD,
116 kD, and 232 kD respectively. FIGS. 7C-D show that dimeric and
monomeric GAC mutants are inactive in the presence and absence of
inorganic phosphate. FIG. 7C-D are graphs of
concentration-dependent enzymatic activities of WT GAC, dimeric GAC
(D391K), and monomeric GAC (D391K, K316E, R459E), without addition
of phosphate (FIG. 7C) and with the addition of 100 mM phosphate
(FIG. 7D). Activities were measured in a 2-step end-point activity
assay where GAC was incubated in the presence of glutamine for 2
minutes at concentrations under 250 nM GAC, and for 30 seconds at
concentrations above 250 nM GAC. Points represent the average
(.+-.SD) of 3 independent experiments. FIG. 7E shows FRET assays
upon addition of 200 nM WT QSY9-labeled GAC (a), the dimeric
QSY9-GAC (D391K) (b), and monomeric QSY9-GAC (K316E, D391K, R459E)
(c) to 20 nM WT 488-labeled GAC. FIG. 7F shows 968 binding
monitored by its quenching of the fluorescence of WT 488-labeled
GAC, dimeric 488-GAC (D391K), and the monomeric GAC (K316E, D391K,
R459E) (10 nM total monomer in each sample). Data points represent
the mean (.+-.SD) of three independent experiments, and were fit as
in FIG. 5C. FIG. 7G shows in vitro inhibition curves of 50 nM
(closed circles) and 5 nM WT GAC (open circles) pre-incubated with
increasing concentrations of 968. Data points represent the mean
(.+-.SD) of three independent experiments, and were fit to a
logistic four parameter curve. Overlaid is the dose dependent
inhibition by 968 of Dbl-induced focus formation (triangles).
[0042] FIGS. 8A-C are representative NMRs of synthesized compounds
with a 968-like scaffold (FIGS. 8A-B) or an SU-11-like scaffold
(FIG. 8C).
[0043] FIG. 9 shows the structure of GLS1 inhibitors SU-1 to SU-36
and depicts their IC.sub.50 values as determined through an in
vitro inhibition assay.
[0044] FIGS. 10A-AJ are graphs showing in vitro quenching and
inhibition of GAC for compounds 968, SU-1, SU-2, SU-4, SU-5, SU-7,
SU-8, SU-10-SU-36, 031, and 27 respectively.
[0045] FIG. 11 is a histogram showing IC.sub.50s from FIG. 9 ranked
in order of potency, including as compared to compound 968.
[0046] FIG. 12 shows the correlation between IC.sub.50 and K.sub.D
of the GLS1 inhibitors.
[0047] FIGS. 13A-F are graphs showing in vitro quenching and
inhibition of 488-KGA for compounds 968, SU-11, SU-14, SU-21,
SU-23, and 27, respectively.
[0048] FIG. 14 is a chart showing the IC.sub.50 values of 968 and
SU-1 to SU-15 for the inhibition of cell growth in a proliferation
assay using the breast cancer cell line MDA-MB-231.
[0049] FIG. 15 shows the inhibition of various compounds in a
proliferation assay in MDA-MB-231 breast cancer cells.
[0050] FIGS. 16A-B are graphs of SU-22 quenching and inhibition of
488-GAC following 30 s UV exposure (FIG. 16A) or after 7 minutes of
binding with or without UV exposure (FIG. 16B).
[0051] FIG. 17 shows SU-22 photo cross linking to GAC in vitro
separated using SDS-PAGE and visualized under UV light.
[0052] FIGS. 18A-D relate to the purification of SU-22 photo cross
linked to GAC in vitro analyzed using gel filtration chromatography
and high performance liquid chromatography of peptide fragments
following reaction with trypsin. FIG. 18A (top) illustrates the
fluorescence of each fraction of the purification protocol for
cross linking SU-22 to the K316E/D391K/R459E GAC mutant.
Additionally, the total protein in each fraction was visualized
using Coomassie blue staining (FIG. 18A (bottom)), further
illustrating the purification of the SU-22 labeled species. FIG.
18B is the absorbance trace showing the isolated SU-22 conjugate
analyzed using analytical gel filtration. The absorbance trace at
280 nm represents the elution of the protein species and the
absorbance trace at 350 nm represents the absorbance of the small
molecule, SU-22. FIG. 18C shows the absorbance profile of the
isolated SU-22-WT GAC conjugate analyzed using UV-vis spectroscopy,
where the absorbance at 350 nm is characteristic of the small
molecule SU-22. FIG. 18D shows the HPLC profile of the SU-22-WT GAC
conjugate, where the absorbance trace at 254 nm represents any
eluted peptides, and the absorbance at 350 nm represents the small
molecule SU-22 conjugated peptide (arrow).
[0053] FIG. 19 shows confocal microscopy images of fixed
Dbl-transformed MEFs following UV stimulation, showing the
subcellular localization of the fluorescent 968 derivative, SU-22,
cross linked in Dbl transformed cells.
[0054] FIG. 20 shows SDS-PAGE gels relating to the isolation of the
photo cross linker, SU-22, cross linked to glutaminase proteins
following UV exposure.
[0055] FIG. 21 relates to the isolation of the photo cross linker,
SU-34, cross linked to glutaminase proteins following UV exposure
and subsequent copper catalyzed click chemistry to attach the
highly fluorescent probe, alexa-488 azide.
DETAILED DESCRIPTION OF THE INVENTION
[0056] One aspect of the present invention relates to a compound,
or a pharmaceutically acceptable salt, ester, enol ether, enol
ester, solvate, hydrate, or prodrug thereof, wherein the compound
is a compound of Formula I:
##STR00005## [0057] wherein: [0058] R is selected from the group
consisting of monocyclic or bicyclic aryl, monocyclic or bicyclic
heteroaryl, and monocyclic or bicyclic heterocyclyl, wherein each
monocyclic or bicyclic aryl, monocyclic or bicyclic heteroaryl, and
monocyclic or bicyclic heterocyclyl can be optionally substituted
from 1 to 4 times with substituents independently selected at each
occurrence thereof from the group consisting of H, halogen,
C.sub.1-6 alkyl, aryl, --OR.sup.8, --CF.sub.3, and --CHF.sub.2;
[0059] R.sup.1 and R.sup.2 are each independently selected from the
group consisting of H, halogen, and C.sub.1-6 alkyl; or R.sup.1 and
R.sup.2 are combined to form .dbd.O; [0060] R.sup.3--R.sup.7 are
each independently selected from the group consisting of H,
halogen, --NO.sub.2, --NR.sup.8R.sup.9, --SO.sub.2NR.sup.8R.sup.9,
--N.sub.3, --C(O)R.sup.8, aryl, heteroaryl, heterocyclyl.
##STR00006##
[0060] and [0061] R.sup.8 and R.sup.9 are each independently
selected from the group consisting of H, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, and aryl; or R.sup.8 and R.sup.9 are
combined with the nitrogen to which they are attached to form a
heterocyclyl, wherein the heterocyclyl can be optionally
substituted with --COOH or --COOMe; and [0062] wherein the compound
is optionally modified to include a tag and/or an attachment to a
solid surface.
[0063] Another aspect of the present invention relates to a
compound, or a pharmaceutically acceptable salt, ester, enol ether,
enol ester, solvate, hydrate, or prodrug thereof, wherein the
compound is a compound of Formula II:
##STR00007## [0064] wherein: [0065] the dotted circle identifies an
active moiety; [0066] X is independently --CR.sub.14a-- or --N;
[0067] R.sub.1a is independently H, --OH, --OR.sub.14a,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, R.sub.14aC(O)--, R.sub.14aOC(O), R.sub.14aS(O)--, or
R.sub.14aS(O).sub.2--; [0068] R.sub.2a, R.sup.3a, R.sub.4a,
R.sub.5a, and R.sub.6a are each independently a photoreactive
moiety, H, halogen, --NO.sub.2, --OH, --OR.sub.14a, --SR.sub.14a,
--NH.sub.2, --NHR.sub.14a, --NR.sub.14aR.sub.15a, R.sub.14aC(O)--,
R.sub.14aOC(O)--, R.sub.14aC(O)O--, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6
alkyl, mono or polycyclic aryl, or mono or polycyclic heteroaryl
with each cyclic unit containing from 1 to 5 heteroatoms selected
from the group consisting of nitrogen, sulfur, and oxygen, wherein
the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl,
arylalkyl, mono or polycyclic aryl, and mono or polycyclic
heteroaryl are optionally substituted with a photoreactive moiety;
or R.sub.2a and R.sub.3a, R.sub.3a and R.sub.4a, R.sub.4a and
R.sub.5a, or R.sub.5a and R.sub.6a are combined to form a
heterocyclic ring optionally substituted with a photoreactive
moiety; [0069] R.sub.7a, R.sub.8a, R.sub.9a, and R.sub.10a are each
independently a photoreactive moiety, H, --OH, --NH.sub.2,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.4-C.sub.7
cycloalkylalkyl, aryl C.sub.6 alkyl, mono or polycyclic aryl, or
mono or polycyclic heteroaryl with each cyclic unit containing from
1 to 5 heteroatoms selected from the group consisting of nitrogen,
sulfur, and oxygen, wherein the aryl, heteroaryl, and aryl
C.sub.1-C.sub.6 alkyl are optionally substituted from 1 to 3 times
with substituents selected from the group consisting of, halogen,
--OH, --NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.1-C.sub.6 alkoxy, --SH, and C.sub.1-C.sub.6 thioalkyl, and
wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl,
arylalkyl, mono or polycyclic aryl, and mono or polycyclic
heteroaryl are optionally substituted with a photoreactive moiety;
and [0070] R.sub.11a, R.sub.12a, R.sub.13a, R.sub.14a, R.sub.15a,
R.sub.16a, and R.sub.17a are each independently a photoreactive
moiety, H, halogen, --OH, --NO.sub.2, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, aryl C.sub.1-C.sub.6
alkyl, mono or polycyclic aryl, wherein the alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkylalkyl, arylalkyl, and mono or
polycyclic aryl are optionally substituted with a photoreactive
moiety and each one of R.sub.11a-R.sub.17a is optionally
substituted with --NH.sub.2, --OH, halogen, --COOH, --NO.sub.2, and
--CN; [0071] wherein the compound comprises at least one
photoreactive moiety; and [0072] wherein the compound is optionally
modified to include a tag and/or an attachment to a solid
surface.
[0073] Another aspect of the present invention relates to a
compound, or a pharmaceutically acceptable salt, ester, enol ether,
enol ester, solvate, hydrate, or prodrug thereof, wherein the
compound is a compound of Formula III:
##STR00008## [0074] wherein: [0075] R is selected from the group
consisting of monocyclic or bicyclic aryl, monocyclic or bicyclic
heteroaryl, and monocyclic or bicyclic heterocyclyl, wherein each
monocyclic or bicyclic aryl, monocyclic or bicyclic heteroaryl, and
monocyclic or bicyclic heterocyclyl can be optionally substituted
from 1 to 4 times with substituents independently selected at each
occurrence thereof from the group consisting of H, halogen,
C.sub.1-6 alkyl, aryl, --OR.sup.8, --CF.sub.3, and --CHF.sub.2;
[0076] R.sup.1 and R.sup.2 are each independently selected from the
group consisting of a photoreactive moiety, H, halogen, and
C.sub.1-6 alkyl optionally substituted with a photoreactive moiety;
or R.sup.1 and R.sup.2 are combined to form .dbd.O; [0077]
R.sup.3-R.sup.7 are each independently selected from the group
consisting of a photoreactive moiety, H, halogen, --NO.sub.2,
--NR.sup.8R.sup.9, --SO.sub.2NR.sup.8R.sup.9, --N.sub.3,
--C(O)R.sup.8, aryl, heteroaryl, and heterocyclyl, wherein the aryl
and heteroaryl are optionally substituted with a photoreactive
moiety; and [0078] R.sup.8 and R.sup.9 are each independently
selected from the group consisting of a photoreactive moiety, H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, and aryl,
wherein the alkyl, alkenyl, alkynyl, and aryl are optionally
substituted with a photoreactive moiety; or R.sup.8 and R.sup.9 are
combined with the nitrogen to which they are attached to form a
heterocyclyl, wherein the heterocyclyl can be optionally
substituted with a photoreactive moiety, --COOH, or --COOMe; [0079]
wherein the compound comprises at least one photoreactive moiety;
and [0080] wherein the compound is optionally modified to include a
tag and/or an attachment to a solid surface.
[0081] The term "halo" or "halogen" means fluoro, chloro, bromo, or
iodo.
[0082] The term "optionally substituted" indicates that a group may
have a substituent at each substitutable atom of the group
(including more than one substituent on a single atom), and the
identity of each substituent is independent of the others.
[0083] The term "substituted" or "substitution" of an atom means
that one or more hydrogen on the designated atom is replaced with a
selection from the indicated group, provided that the designated
atom's normal valency is not exceeded. "Unsubstituted" atoms bear
all of the hydrogen atoms dictated by their valency. When a
substituent is oxo (i.e., .dbd.O), then 2 hydrogens on the atom are
replaced. Combinations of substituents and/or variables are
permissible only if such combinations result in stable compounds;
by "stable compound" or "stable structure" is meant a compound that
is sufficiently robust to survive isolation to a useful degree of
purity from a reaction mixture, and formulation into an efficacious
therapeutic agent. Exemplary substitutents include, without
limitation, oxo, thio (i.e. .dbd.S), nitro, cyano, halo, OH,
NH.sub.2, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.4-C.sub.7 cycloalkylalkyl, monocyclic aryl,
monocyclic hetereoaryl, polycyclic aryl, and polycyclic
heteroaryl.
[0084] The term "monocyclic" indicates a molecular structure having
one ring.
[0085] The term "polycyclic" indicates a molecular structure having
two ("bicyclic") or more rings, including, but not limited to,
fused, bridged, or spiro rings.
[0086] The term "alkyl" means an aliphatic hydrocarbon group which
may be straight or branched having about 1 to about 6 carbon atoms
in the chain. Branched means that one or more lower alkyl groups
such as methyl, ethyl or propyl are attached to a linear alkyl
chain. Exemplary alkyl groups include methyl, ethyl, n-propyl,
i-propyl, n-butyl, t-butyl, n-pentyl, and 3-pentyl.
[0087] The term "thioalkyl" means an alkyl group as described above
bonded through a sulfur linkage.
[0088] The term "alkenyl" means an aliphatic hydrocarbon group
containing a carbon-carbon double bond and which may be straight or
branched having about 2 to about 6 carbon atoms in the chain.
Preferred alkenyl groups have 2 to about 4 carbon atoms in the
chain. Branched means that one or more lower alkyl groups such as
methyl, ethyl, or propyl are attached to a linear alkenyl chain.
Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, and
i-butenyl.
[0089] The term "alkynyl" means an aliphatic hydrocarbon group
containing a carbon-carbon triple bond and which may be straight or
branched having about 2 to about 6 carbon atoms in the chain.
Preferred alkynyl groups have 2 to about 4 carbon atoms in the
chain. Branched means that one or more lower alkyl groups such as
methyl, ethyl, or propyl are attached to a linear alkynyl chain.
Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl,
2-butynyl, 3-methylbutynyl, and n-pentynyl.
[0090] The term "alkoxy" means an alkyl-O--, alkenyl-O--, or
alkynyl-O-- group wherein the alkyl, alkenyl, or alkynyl group is
described above. Exemplary alkoxy groups include methoxy, ethoxy,
n-propoxy, i-propoxy, n-butoxy, pentoxy, and hexoxy.
[0091] The term "cycloalkyl" refers to a non-aromatic saturated or
unsaturated mono- or polycyclic ring system which may contain 3 to
6 carbon atoms; and which may include at least one double bond.
Exemplary cycloalkyl groups include, without limitation,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropenyl,
cyclobutenyl, cyclopentenyl, cyclohexenyl, anti-bicyclopropane, and
syn-bicyclopropane.
[0092] The term "cycloalkylalkyl" refers to a radical of the
formula --R.sup.aR.sup.b where R.sup.a is an alkyl radical as
defined above and R.sup.b is a cycloalkyl radical as defined above.
The alkyl radical and the cycloalkyl radical may be optionally
substituted as defined above.
[0093] The term "aryl" refers to aromatic monocyclic or polycyclic
ring system containing from 6 to 19 carbon atoms, where the ring
system may be optionally substituted. Aryl groups of the present
invention include, but are not limited to, groups such as phenyl,
naphthyl, azulenyl, phenanthrenyl, anthracenyl, fluorenyl, pyrenyl,
triphenylenyl, chrysenyl, and naphthacenyl.
[0094] The term "arylalkyl" refers to a radical of the formula
--R.sup.aR.sup.b where R.sup.a is an alkyl radical as defined above
and R.sup.b is an aryl radical as defined above. The alkyl radical
and the cycloalkyl radical may be optionally substituted as defined
above.
[0095] The term "arylarylalkyl" refers to a radical of the formula
--R.sup.aR.sup.bR.sup.c where R.sup.a is an alkyl as defined above,
R.sup.b is an aryl radical as defined above, and R.sup.c is an aryl
radical as defined above. The alkyl radical and both aryl radicals
may be optionally substituted as defined above.
[0096] The term "heterocyclyl" refers to a stable 3- to 18-membered
ring radical which consists of carbon atoms and from one to five
heteroatoms selected from the group consisting of nitrogen, oxygen
and sulfur. For purposes of this invention, the heterocyclyl
radical may be a monocyclic, or a polycyclic ring system, which may
include fused, bridged, or spiro ring systems; and the nitrogen,
carbon, or sulfur atoms in the heterocyclyl radical may be
optionally oxidized; the nitrogen atom may be optionally
quaternized; and the ring radical may be partially or fully
saturated. Examples of such heterocyclyl radicals include, without
limitation, azepinyl, azocanyl, pyranyl dioxanyl, dithianyl,
1,3-dioxolanyl, tetrahydrofuryl, dihydropyrrolidinyl,
decahydroisoquinolyl, imidazolidinyl, isothiazolidinyl,
isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,
2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl,
2-oxoazepinyl, oxazolidinyl, oxiranyl, piperidinyl, piperazinyl,
4-piperidonyl, pyrrolidinyl, pyrazolidinyl, thiazolidinyl,
tetrahydropyranyl, thiamorpholinyl, thiamorpholinyl sulfoxide, and
thiamorpholinyl sulfone.
[0097] The term "heteroaryl" refers to an aromatic ring radical
which consists of carbon atoms and from one to five heteroatoms
selected from the group consisting of nitrogen, oxygen, and sulfur.
For purposes of this invention the heteroaryl may be a monocyclic
or polycyclic ring system; and the nitrogen, carbon, and sulfur
atoms in the heteroaryl ring may be optionally oxidized; the
nitrogen may optionally be quaternized. Examples of heteroaryl
groups include, without limitation, pyrrolyl, pyrazolyl,
imidazolyl, triazolyl, furyl, thiophenyl, oxazolyl, isoxazolyl,
thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridyl,
pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thienopyrrolyl,
furopyrrolyl, indolyl, azaindolyl, isoindolyl, indolinyl,
indolizinyl, indazolyl, benzimidazolyl, imidazopyridinyl,
benzotriazolyl, benzoxazolyl, benzoxadiazolyl, benzothiazolyl,
pyrazolopyridinyl, triazolopyridinyl, thienopyridinyl,
benzothiadiazolyl, benzofuyl, benzothiophenyl, quinolinyl,
isoquinolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl,
cinnolinyl, quinazolinyl, quinolizilinyl, phthalazinyl,
benzotriazinyl, chromenyl, naphthyridinyl, acrydinyl, phenanzinyl,
phenothiazinyl, phenoxazinyl, pteridinyl, and purinyl.
[0098] Further heterocycles and heteroaryls are described in
COMPREHENSIVE HETEROCYCLIC CHEMISTRY: THE STRUCTURE, REACTIONS,
SYNTHESIS AND USE OF HETEROCYCLIC COMPOUNDS Vol. 1-8 (Alan R.
Katritzky et al. eds., 1.sup.st ed. 1984), which is hereby
incorporated by reference in its entirety.
[0099] A "photoreactive moiety" as used herein is a moiety that
becomes reactive when exposed to ultraviolet or visible light.
Photoreactive moieties for use in the compounds of Formula II and
Formula III include, for example, aryl azides, diazirines, and
benzophenone.
[0100] The compounds of the present invention (or pharmaceutically
acceptable salts, esters, enol ethers, enol esters, solvates,
hydrates, or prodrugs thereof) can optionally be modified to
include a tag. A "tag" as used herein includes any labeling moiety
that facilitates the detection, quantitation, isolation, and/or
purification of a compound (i.e., a compound of the present
invention, a compound-glutaminase GLS1 protein conjugate as
described infra, a conjugated compound/inhibitor as described
infra, and/or a conjugated glutaminase GLS1 protein as described
infra). Methods for modifying small molecules to include tags are
well known in the art. For example, click chemistry (see, e.g.,
U.S. Pat. No. 7,375,234 to Sharpless et al., which is hereby
incorporated by reference in its entirety) may be used to attach a
tag to a compound.
[0101] Suitable tags include purification tags, radioactive or
fluorescent labels, enzymatic tags, prosthetic groups, luminescent
materials, bioluminescent materials, positron emitting metals,
nonradioactive paramagnetic metal ions, and any other signal
suitable for detection and/or measurement by radiometric,
colorimetric, fluorometric, size-separation, or precipitation
means, or other means known in the art.
[0102] Purification tags, such as maltose-binding protein (MBP-),
poly-histidine (His.sub.6-), or a glutathione-S-transferase (GST-),
can assist in compound purification or separation but can later be
removed, i.e., cleaved from the compound following recovery.
Protease-specific cleavage sites can be used to facilitate the
removal of the purification tag. The desired product can be
purified further to remove the cleaved purification tags.
[0103] Other suitable tags include radioactive labels, such as,
.sup.125I, .sup.123I, .sup.131I, .sup.111In, .sup.112In,
.sup.113In, .sup.115In, .sup.99TC, .sup.213Bi, .sup.14C, .sup.51Cr,
.sup.153Gd, .sup.159Gd, .sup.68Ga, .sup.67Ga, .sup.68Ge,
.sup.166Ho, .sup.140La, .sup.177Lu, .sup.54Mn, .sup.99Mo,
.sup.103Pd, .sup.32P, .sup.142Pr, .sup.149Pm, .sup.186Re,
.sup.188Re, .sup.105Rh, .sup.97Ru, .sup.153Sm, .sup.47Sc,
.sup.75Se, .sup.85Sr, .sup.35S, .sup.201Ti, .sup.113Sn, .sup.117Sn,
.sup.3H, .sup.133Xe, .sup.169Yb, .sup.175Yb, .sup.90Y, and
.sup.65Zn. Methods of radiolabeling compounds are known in the art
and described in U.S. Pat. No. 5,830,431 to Srinivasan et al.,
which is hereby incorporated by reference in its entirety.
Radioactivity is detected and quantified using a scintillation
counter or autoradiography. Further examples include positron
emitting metals using various positron emission tomographies, and
nonradioactive paramagnetic metal ions.
[0104] Alternatively, the compound can be conjugated to a
fluorescent tag. Suitable fluorescent tags include, without
limitation, chelates (europium chelates), fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, Lissamine,
phycoerythrin, Texas Red, and umbelliferone. The fluorescent labels
can be conjugated to the compounds using techniques disclosed in
CURRENT PROTOCOLS IN IMMUNOLOGY (Coligen et al. eds., 1991), which
is hereby incorporated by reference in its entirety. Fluorescence
can be detected and quantified using a fluorometer.
[0105] Enzymatic tags generally catalyze a chemical alteration of a
chromogenic substrate which can be measured using various
techniques. For example, the enzyme may catalyze a color change in
a substrate, which can be measured spectrophotometrically.
Alternatively, the enzyme may alter the fluorescence or
chemiluminescence of the substrate. Examples of suitable enzymatic
tags include luciferases (e.g., firefly luciferase and bacterial
luciferase; see e.g., U.S. Pat. No. 4,737,456 to Weng et al., which
is hereby incorporated by reference in its entirety), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidases (e.g., horseradish peroxidase), alkaline phosphatase,
0-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,
glucose oxidase, galactose oxidase, and glucose-6-phosphate
dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine
oxidase), lactoperoxidase, microperoxidase, and the like.
Techniques for conjugating enzymes to proteins and peptides are
described in O'Sullivan et al., Methods for the Preparation of
Enzyme--Antibody Conjugates for Use in Enzyme Immunoassay, in
METHODS IN ENZYMOLOGY 147-66 (Langone et al. eds., 1981), which is
hereby incorporated by reference in its entirety.
[0106] Prosthetic group complexes include, but are not limited to,
streptavidin/biotin and avidin/biotin. Alternatively, the compound
can be conjugated to a luminescent or bioluminescent material
including, but not limited to, luminol, luciferase, luciferin, and
aequorin.
[0107] The compounds of the present invention (or pharmaceutically
acceptable salts, esters, enol ethers, enol esters, solvates,
hydrates, or prodrugs thereof) can optionally be modified to
include an attachment to a solid surface, such as a fibrous test
strip, a column, a multi-well microliter plate, a test tube, or
beads. Methods for attaching small molecules to such surfaces,
including covalent attachment (for example via click chemistry, as
described supra) as well as non-covalent attachment through the use
of antibody-antigen partners, complementary nucleic acids, etc.,
are well known in the art.
[0108] Pharmaceutically acceptable salts include, but are not
limited to, amine salts, such as but not limited to, N,
N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia,
diethanolamine and other hydroxyalkylamines, ethylenediamine,
N-methylglucamine, procaine, N-benzylphenethylamine,
1-para-chlorobenzyl-2-pyrrolidin-1'-ylmethyl-benzimidazole,
diethylamine and other alkylamines, piperazine, and tris
(hydroxymethyl) aminomethane; alkali metal salts, such as but not
limited to, lithium, potassium, and sodium; alkali earth metal
salts, such as but not limited to, barium, calcium, and magnesium;
transition metal salts, such as but not limited to, zinc; and other
metal salts, such as but not limited to, sodium hydrogen phosphate
and disodium phosphate; and also including, but not limited to,
salts of mineral acids, such as but not limited to, hydrochlorides
and sulfates; and salts of organic acids, such as but not limited
to, acetates, lactates, malates, tartrates, citrates, ascorbates,
succinates, butyrates, valerates and fumarates. Pharmaceutically
acceptable esters include, but are not limited to, alkyl, alkenyl,
alkynyl, aryl, heteroaryl, cycloalkyl and heterocyclyl esters of
acidic groups, including, but not limited to, carboxylic acids,
phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids,
and boronic acids. Pharmaceutical acceptable enol ethers include,
but are not limited to, derivatives of formula C.dbd.C (OR) where R
is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,
or heterocyclyl. Pharmaceutically acceptable enol esters include,
but are not limited to, derivatives of formula C.dbd.C (OC(O) R)
where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
cycloalkyl, or heterocyclyl. Pharmaceutical acceptable solvates and
hydrates are complexes of a compound with one or more solvent or
water molecules, or 1 to about 100, or 1 to about 10, or one to
about 2, 3 or 4, solvent or water molecules.
[0109] Exemplary Formula I compounds of the present invention
include, without limitation, any of the following GLS1 inhibitors
(and pharmaceutically acceptable salts or produgs thereof, and
optionally modified to include a tag and/or an attachment to a
solid surface).
##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013##
##STR00014## ##STR00015##
[0110] In a preferred embodiment, the compound of Formula I is
selected from the group consisting of SU-11, SU-14, SU-16, SU-17,
SU-18, SU-19, SU-20, SU-21, SU-22, SU-23, SU-24, SU-25, SU-26,
SU-27, SU-28, SU-29, SU-30, SU-31, SU-32, SU-33, SU-34, SU-35, and
SU-36 (and pharmaceutically acceptable salts or produgs thereof,
and optionally modified to include a tag and/or an attachment to a
solid surface).
[0111] In a preferred embodiment, the compound of Formula I is
selected from the group consisting of SU-11, SU-16, SU-17, SU-19,
SU-20, SU-23, SU-24, SU-26, SU-27, SU-28, SU-29, SU-30, SU-31,
SU-32, SU-35, and SU-36 (and pharmaceutically acceptable salts or
produgs thereof, and optionally modified to include a tag and/or an
attachment to a solid surface).
[0112] Exemplary Formula II compounds of the present invention
include any of the glutaminase inhibitors disclosed in
International Patent Application No. PCT/US10/28688, which is
hereby incorporated by reference in its entirety, but being
modified to include a photoreactive moiety in accordance with
Formula II herein (and pharmaceutically acceptable salts or produgs
thereof, and optionally modified to include a tag and/or an
attachment to a solid surface).
[0113] With respect to compounds of Formula II, suitable active
moieties are described in International Patent Application No.
PCT/US10/28688, which is hereby incorporated by reference in its
entirety, and include the active moieties shown below (optionally
modified to include a photoreactive moiety, a tag, and/or an
attachment to a solid surface).
##STR00016## ##STR00017##
In a preferred embodiment, the active moiety is
##STR00018##
[0114] Exemplary Formula III compounds of the present invention
include SU-21, SU-22, SU-23, SU-24, SU-33, and SU-34 (and
pharmaceutically acceptable salts or produgs thereof, and
optionally modified to include a tag and/or an attachment to a
solid surface).
[0115] As noted above, photoreactive moieties for use in the
compounds of Formula II and Formula III include, for example, aryl
azides, diazirines, and benzophenone. Suitable examples include,
without limitation, --N.dbd.N.sup.+.dbd.N.sup.-;
##STR00019##
As will be apparent to the skilled artisan, aryl azides and
benzophenone are attached to an aromatic ring. Thus, when the
photoreactive moiety is an aryl azide or benzophenone, it is
present in Formula II at the R.sub.2a--R.sub.6a, R.sub.11a,
R.sub.12a, R.sub.13a. R.sub.16a, or R.sub.17a position and is
present in Formula III at the R.sub.3--R.sub.7 positions.
[0116] With respect to Formula II compounds, compounds having a
photoreactive moiety in the R.sub.2a--R.sub.6a ring have been shown
to be effective. Inhibition assays using 968-like compounds have
shown that the R.sub.7a--R.sub.10a ring can tolerate many different
substitutions while maintaining efficacy. Thus, it is expected that
the photoreactive moiety may be located on this ring as well. In at
least one embodiment, the photoreactive moiety is present at the
R.sub.4a position.
[0117] With respect to Formula III compounds, compounds having a
photoreactive moiety at the R.sup.5 position have been shown to be
effective. Inhibition assays using compounds of Formula I, which
are structurally similar to those of Formula III, have shown that
such compounds can tolerate many different substitutions at the
R.sup.1-R.sup.4, R.sup.6, and R.sup.7 positions while maintaining
efficacy. Thus, it is expected that the photoreactive moiety may be
located at these positions as well. In at least one embodiment, the
photoreactive moiety is present at the R.sup.5 position.
[0118] Another aspect of the present invention relates to a
pharmaceutical composition comprising a compound of Formula I,
Formula II, or Formula III, or a pharmaceutically acceptable salt,
ester, enol ether, enol ester, solvate, hydrate, or prodrug
thereof.
[0119] According to this aspect of the present invention, the
pharmaceutical compositions can comprise a compound of the present
invention and a pharmaceutically acceptable carrier and,
optionally, one or more additional active agent(s) as discussed
below.
[0120] Numerous standard references are available that describe
procedures for preparing various formulations suitable for
administering the compounds according to the invention. Examples of
potential formulations and preparations are contained, for example,
in the HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (American
Pharmaceutical Association, current edition), PHARMACEUTICAL DOSAGE
FORMS: TABLETS (Lieberman et al. eds., Marcel Dekker, Inc., pubs.,
current edition), and REMINGTON'S PHARMACEUTICAL SCIENCES 1553-93
(Arthur Osol ed., current edition), which are hereby incorporated
by reference in their entirety.
[0121] Any pharmaceutically acceptable liquid carrier suitable for
preparing solutions, suspensions, emulsions, syrups and elixirs may
be employed in the composition of the invention. Compounds of the
present invention may be dissolved or suspended in a
pharmaceutically acceptable liquid carrier such as water, an
organic solvent, or a pharmaceutically acceptable oil or fat, or a
mixture thereof. The liquid composition may contain other suitable
pharmaceutical additives such as solubilizers, emulsifiers,
buffers, preservatives, sweeteners, flavoring agents, suspending
agents, thickening agents, coloring agents, viscosity regulators,
stabilizers, osmo-regulators, or the like. Examples of liquid
carriers suitable for oral and parenteral administration include
water (particularly containing additives as above, e.g., cellulose
derivatives, preferably sodium carboxymethyl cellulose solution),
alcohols (including monohydric alcohols and polyhydric alcohols,
e.g., glycols) or their derivatives, or oils (e.g., fractionated
coconut oil and arachis oil). For parenteral administration the
carrier may also be an oily ester such as ethyl oleate or isopropyl
myristate.
[0122] It will be understood that the specific dose level for any
particular patient will depend upon a variety of factors, including
the activity of the specific compound employed, the age, body
weight, general health, sex, diet time of administration, route of
administration, rate of excretion, drug combination and the
severity of the particular disease undergoing therapy.
[0123] Another aspect of the present invention relates to a method
of reducing the production of glutamate from glutamine by
glutaminase GLS1 in a sample. This method involves inhibiting
glutaminase GLS1 activity in the sample by providing a compound and
contacting glutaminase GLS1 in the sample with the compound to
reduce the production of glutamate from glutamine in the sample,
where the compound is a compound of Formula I, Formula II, or
Formula III, or a pharmaceutically acceptable salt, ester, enol
ether, enol ester, solvate, hydrate, or prodrug thereof.
[0124] The term "reduce" means to suppress, decrease, diminish, or
lower the production of glutamate from glutamine.
[0125] Suitable samples include those described infra.
[0126] In all aspects of the present invention directed to methods
involving contacting a sample with one or more compounds,
contacting can be carried out using methods that will be apparent
to the skilled artisan, and can be done in vitro, ex vivo, or in
vivo.
[0127] Compounds of the present invention may be delivered directly
to a targeted cell/tissue/organ. Additionally and/or alternatively,
the compounds may be administered to a non-targeted area along with
one or more agents that facilitate migration of the compounds to
(and/or uptake by) a targeted tissue, organ, or cell. As will be
apparent to one of ordinary skill in the art, the compound itself
can be modified to facilitate its transport to a target tissue,
organ, or cell, including its transport across the blood-brain
barrier; and/or to facilitate its uptake by a target cell (e.g.,
its transport across cell membranes).
[0128] In vivo administration can be accomplished either via
systemic administration to the subject or via targeted
administration to affected tissues, organs, and/or cells, as
described above. Typically, the therapeutic agent (i.e., a compound
of the present invention) will be administered to a patient in a
vehicle that delivers the therapeutic agent(s) to the target cell,
tissue, or organ. Typically, the therapeutic agent will be
administered as a pharmaceutical formulation, such as those
described above.
[0129] The compounds of the present invention can be administered,
e.g., by intravenous injection, intramuscular injection,
subcutaneous injection, intraperitoneal injection, topical
application, sublingual, intraarticular (in the joints),
intradermal, buccal, ophthalmic (including intraocular),
intranasally (including using a cannula), or by other routes. The
compounds of formulae I, II, and/or III (as well as compounds
comprising their active moieties) can be administered orally, e.g.,
as a tablet or cachet containing a predetermined amount of the
active ingredient, gel, pellet, paste, syrup, bolus, electuary,
slurry, capsule, powder, granules, as a solution or a suspension in
an aqueous liquid or a non-aqueous liquid, as an oil-in-water
liquid emulsion or a water-in-oil liquid emulsion, via a micellar
formulation (see, e.g. WO 97/11682, which is hereby incorporated by
reference in its entirety) via a liposomal formulation (see, e.g.,
European Patent No. 736299, WO 99/59550, and WO 97/13500, which are
hereby incorporated by reference in their entirety), via
formulations described in WO 03/094886, which is hereby
incorporated by reference in its entirety, or in some other form.
The compounds of the present invention can also be administered
transdermally (i.e. via reservoir-type or matrix-type patches,
microneedles, thermal poration, hypodermic needles, iontophoresis,
electroporation, ultrasound or other forms of sonophoresis, jet
injection, or a combination of any of the preceding methods
(Prausnitz et al., Nature Reviews Drug Discovery 3:115 (2004),
which is hereby incorporated by reference in its entirety). The
compounds can be administered locally, for example, at the site of
injury to an injured blood vessel. The compounds can be coated on a
stent. The compounds can be administered using high-velocity
transdermal particle injection techniques using the hydrogel
particle formulation described in U.S. Patent Publication No.
20020061336, which is hereby incorporated by reference in its
entirety. Additional particle formulations are described in WO
00/45792, WO 00/53160, and WO 02/19989, which are hereby
incorporated by reference in their entirety. An example of a
transdermal formulation containing plaster and the absorption
promoter dimethylisosorbide can be found in WO 89/04179, which is
hereby incorporated by reference in its entirety. WO 96/11705,
which is hereby incorporated by reference in its entirety, provides
formulations suitable for transdermal administration.
[0130] For use as aerosols, a compound of the present invention in
solution or suspension may be packaged in a pressurized aerosol
container together with suitable propellants, for example,
hydrocarbon propellants like propane, butane, or isobutane with
conventional adjuvants. The compounds of the present invention also
may be administered in a non-pressurized form.
[0131] Exemplary delivery devices include, without limitation,
nebulizers, atomizers, liposomes (including both active and passive
drug delivery techniques) (Wang & Huang, "pH-Sensitive
Immunoliposomes Mediate Target-Cell-Specific Delivery and
Controlled Expression of a Foreign Gene in Mouse," Proc. Nat'l
Acad. Sci. USA 84:7851-55 (1987); Bangham et al., "Diffusion of
Univalent Ions Across the Lamellae of Swollen Phospholipids," J.
Mol. Biol. 13:238-52 (1965); U.S. Pat. No. 5,653,996 to Hsu; U.S.
Pat. No. 5,643,599 to Lee et al.; U.S. Pat. No. 5,885,613 to
Holland et al.; U.S. Pat. No. 5,631,237 to Dzau & Kaneda; U.S.
Pat. No. 5,059,421 to Loughrey et al.; Wolff et al., "The Use of
Monoclonal Anti-Thy1 IgG1 for the Targeting of Liposomes to AKR-A
Cells in Vitro and in Vivo," Biochim. Biophys. Acta 802:259-73
(1984), each of which is hereby incorporated by reference in its
entirety), transdermal patches, implants, implantable or injectable
protein depot compositions, and syringes. Other delivery systems
which are known to those of skill in the art can also be employed
to achieve the desired delivery of the compound to the desired
organ, tissue, or cells in vivo to effect this aspect of the
present invention.
[0132] Contacting (including in vivo administration) can be carried
out as frequently as required and for a duration that is suitable
to provide the desired effect. For example, contacting can be
carried out once or multiple times, and in vivo administration can
be carried out with a single sustained-release dosage formulation
or with multiple (e.g., daily) doses.
[0133] The amount to be administered will, of course, vary
depending upon the particular conditions and treatment regimen. The
amount/dose required to obtain the desired effect may vary
depending on the agent, formulation, cell type, culture conditions
(for ex vivo embodiments), the duration for which treatment is
desired, and, for in vivo embodiments, the individual to whom the
agent is administered.
[0134] Effective amounts can be determined empirically by those of
skill in the art. For example, this may involve assays in which
varying amounts of the compound of the invention are administered
to cells in culture and the concentration effective for obtaining
the desired result is calculated. Determination of effective
amounts for in vivo administration may also involve in vitro assays
in which varying doses of agent are administered to cells in
culture and the concentration of agent effective for achieving the
desired result is determined in order to calculate the
concentration required in vivo. Effective amounts may also be based
on in vivo animal studies.
[0135] The compounds of the present invention can be administered
alone or as an active ingredient of a pharmaceutical formulation,
such as those described above. The compounds of the present
invention can be administered in a form where the active ingredient
is substantially pure.
[0136] Another aspect of the present invention relates to a method
of treating a subject with a condition mediated by production of
glutamate from glutamine. The method involves selecting a subject
with a condition mediated by production of glutamate from glutamine
and administering to said selected subject an inhibitor of GLS1
activity under conditions effective to treat the condition mediated
by production of glutamate from glutamine. In this aspect of the
present invention, the inhibitor of GLS1 activity is a compound of
Formula I, Formula II, or Formula III, or a pharmaceutically
acceptable salt, ester, enol ether, enol ester, solvate, hydrate,
or prodrug thereof.
[0137] The term "treatment" or "treating" means any manner in which
one or more of the symptoms of a disease or disorder are
ameliorated or otherwise beneficially altered. Treatment also
encompasses any pharmaceutical use of the compositions herein, such
as use for treating diseases or disorders mediated by the
production of glutamate from glutamine.
[0138] Splice isoforms, namely kidney-type glutaminase (KGA) and
isoform C (GAC) of the Gls1 gene (gene locus 2q32-q34) represent
the translated forms of the human kidney-type glutaminase or GLS1,
an enzyme found in abundance in proliferating cells, immune cells,
kidney, brain, muscle, and other tissues, and is generally referred
to here throughout as GLS1. Gls1 gene products are involved in the
hydrolysis of glutamine to glutamate and ammonium.
[0139] In one embodiment, this aspect of the present invention can
be carried out by inhibiting overexpression-independent GLS1
activity and/or inhibiting GLS1 activity independent of exogenous
phosphate addition. Alternatively, an activating phosphorylation
event on GLS1 can be inhibited. As a further alternative of the
method of the present invention, inhibition of GLS1 activity can be
performed by inhibiting GLS1 hyperactivity.
[0140] Although GLS1 expression has been found to be increased in
some cancers, applicants have found that the participation of GLS1
is not limited to an increase in expression. Some cancer cells
(such as the breast cancer cell line SKBR3) have been found to
exhibit a particular splice isoform of GLS1 (GAC) expression levels
which are similar to normal cells, but are still dependent on the
presence of GLS1 for cell growth (see, e.g. WO 10/111504, which is
hereby incorporated by reference in its entirety). Thus, by
reducing the normal expression levels of this splice isoform of
GLS1, one can inhibit kidney-type glutaminase activity in cancer
cells.
[0141] GLS1 isolated from cancer cells can show an elevated
glutaminase activity level relative to GLS1 isolated from normal
cells when assayed in the absence of phosphate, but in the presence
of phosphate the enzymes isolated from both normal and cancer cells
show a similar extent of activation per amount of GLS1 (see, e.g.
WO 10/111504, which is hereby incorporated by reference in its
entirety). Thus, the GLS1 in cancer cells is not dependent on the
exogenous addition of phosphate to be active. Inhibition of the
phosphate-independent activation of GLS1 in cancer cells would
inhibit the production of glutamate from glutamine.
[0142] One way in which the GLS1 activity from cancer cells may be
increased relative to the GLS1 activity in normal cells is by a
phosphorylation event that occurs on GLS1. If the phosphorylations
on GLS1 are removed/blocked using either alkaline phosphate or a
small molecule (e.g., compound 968), the ability for GLS1 to
produce glutamate from glutamine is limited.
[0143] The activation state of GLS1 may vary among different cancer
cells, regardless of the expression levels of GLS1. A higher amount
of activity may be referred to as "hyperactivity". For example, Dbl
transformed cells and Cdc42 F28L transformed cells contain similar
levels of GLS1 as do untransformed NIH 3T3 cells. However, the GLS1
in the Dbl and Cdc42 transformed cells shows a higher activation
than in the non-transformed cells, with the GLS1 from the Dbl cells
being approximately twice as active than the GLS1 from the Cdc42
transformed cells (see, e.g. WO 10/111504, which is hereby
incorporated by reference in its entirety). Thus, the GLS1 in the
Dbl transformed cells is hyperactive. Inhibiting the hyperactivity
of GLS1 in Dbl cells would limit the production of glutamate from
glutamine by glutaminase C.
[0144] In a preferred embodiment, the glutaminase GLS1 is any
translated isoform of Gls1 that exhibits measurable glutamine
deamidation activity, i.e. hydrolyses glutamine to produce
glutamate and ammonia. This includes GAC and KGA.
[0145] Glutaminase inhibitors have been shown to have efficacy in
cancers of the breast, brain, lung, ovary, pancreas, colon, and
multiple myeloma. It is expected that GLS1 inhibitors of Formula I,
Formula II, and Formula III of the present application would show
efficacy in these and any other cancer which was glutamine
dependent and/or had high GLS1 expression and/or activation. Thus,
the conditions mediated by production of glutamate from glutamine
according to the present invention include, without limitation, any
cancer that exhibits active glutaminase activity, as the result of
GLS1 expression and/or activation.
[0146] A subject or patient in whom administration of the
therapeutic compound is an effective therapeutic regimen for a
disease or disorder is preferably a human, but can be any animal,
including a laboratory animal in the context of a clinical trial or
screening or activity experiment. Thus, as can be readily
appreciated by one of ordinary skill in the art, the methods,
compounds and compositions of the present invention are
particularly suited to administration to any animal, particularly a
mammal, and including, but by no means limited to, humans, domestic
animals, such as feline (e.g., cats) or canine (e.g., dogs)
subjects, farm animals, such as but not limited to bovine (e.g.,
cows), equine (e.g., horses), caprine (e.g., goats), ovine (e.g.,
sheep), and porcine (e.g., pigs) subjects, wild animals (whether in
the wild or in a zoological garden), research animals, such as
mice, rats, rabbits, guinea pigs, goats, sheep, pigs, dogs, cats,
horses, cows, camels, llamas, monkeys, zebrafish etc., avian
species, such as chickens, turkeys, songbirds, etc., i.e., for
veterinary medical use.
[0147] Administering may be carried out as described supra.
[0148] Another aspect of the present invention relates to methods
that involve the formation of a conjugate between a compound of
Formula II or Formula III and glutaminase GLS1 protein. As will be
apparent to the skilled artisan, inhibitor-glutaminase GLS1 protein
conjugates can be made using photoreactive glutaminase inhibitor
compounds of Formula II and Formula III. Briefly, when the
glutaminase inhibitor is allowed to bind to glutaminase GLS1
protein, covalent modification can be initiated by exposing the
inhibitor to an appropriate light source, thereby forming the
conjugate. The ability to form inhibitor-glutaminase GLS1 protein
conjugates can be utilized for the detection, quantitation,
separation, and/or purification of the inhibitors and/or the
glutaminase GLS1 protein.
[0149] One embodiment of this aspect of the present invention
relates to a method of detecting glutaminase GLS1 protein in a
sample. This method involves providing a sample potentially
containing glutaminase GLS1 protein; contacting the sample with a
compound comprising a photoreactive moiety; exposing the compound
to a light source under conditions effective to form a conjugate
between the compound and glutaminase GLS1 protein, if present in
the sample, through covalent modification of the photoreactive
moiety; and detecting whether any compound-glutaminase GLS1 protein
conjugates are formed, wherein formation of a compound-glutaminase
GLS1 protein conjugate indicates the presence of glutaminase GLS1
protein in the sample; and wherein the compound is a compound of
Formula II or Formula III, or a pharmaceutically acceptable salt,
ester, enol ether, enol ester, solvate, hydrate, or prodrug
thereof.
[0150] Another embodiment of this aspect of the present invention
relates to a method of producing a glutaminase
inhibitor-glutaminase GLS1 protein conjugate in a sample. This
method involves providing a sample containing one of (i)
glutaminase GLS1 protein and (ii) a compound comprising a
photoreactive moiety; contacting the sample with the other of (i)
glutaminase GLS1 protein and (ii) a compound comprising a
photoreactive moiety; and exposing the compound to a light source
under conditions effective to form a conjugate between the compound
and glutaminase GLS1 protein through covalent modification of a
photoreactive moiety; wherein the compound is a compound of Formula
II or Formula III, or a pharmaceutically acceptable salt, ester,
enol ether, enol ester, solvate, hydrate, or prodrug thereof.
[0151] As will be apparent to the skilled artisan, once the
conjugate is produced, further steps may optionally be carried out
to, for example, detect (including imaging), quantitate, isolate,
and/or purify the inhibitor and/or glutaminase GLS1 protein. These
steps may be used, for example, to identify cells/tissue in which
glutaminase GLS1 is present, to determine the subcellular
localization of glutaminase GLS1, to monitor glutaminase GLS1
and/or the inhibitor during cellular fractionation, etc. These
steps can be facilitated by using an inhibitor that has been
appropriately tagged, as described supra.
[0152] Detecting and/or quantitating according to all aspects of
the present invention include detection and/or measurement by
radiometric, colorimetric, fluorometric, size-separation, or
precipitation means, or other means known in the art. By way of
example, radioactivity can be detected and quantified using a
scintillation counter or autoradiography, fluorescence can be
detected and quantified using a fluorometer, color changes
catalyzed by enzymatic tags can be measured spectrophotometrically.
Other methods for detecting and quantifying are well known in the
art.
[0153] As will be apparent to the skilled artisan, this aspect of
the present invention may be carried out in vitro, ex vivo, or in
vivo. Contacting may be carried out as described supra.
[0154] Suitable samples according to this and all aspects of the
present invention include, without limitation, purified proteins,
cells, cell extracts, and tissue. Methods involving cell or tissue
samples may be carried out using a cell or tissue that is present
within a subject, or with a cell or tissue that has been isolated
from a subject. Suitable cells and tissues include those of the
cancers described supra. Suitable subjects in which the cell/tissue
may be contained or from which the cell/tissue may be isolated
include the subjects described supra.
EXAMPLES
[0155] The following examples are provided to illustrate
embodiments of the present invention but are by no means intended
to limit its scope.
Example 1--Isogenic, Dbl-Inducible Cell System Culture Conditions
and Immunofluorescence Staining
[0156] Inducible oncogenic Dbl cell lines were created using the
TET-OFF system in mouse embryonic fibroblasts (MEFs) following the
manufacturer's instructions (Clontech). Briefly, PCR products for
onco-Dbl-containing NotI and XbaI restriction endonuclease sites
were cloned in the per2.1 vector using the TOPO-TA cloning kit
(Invitrogen) and subsequently subcloned into the p-TREHA vector
(Clontech). The pTRE-HA-onco-Dbl was then co-transfected with
pMET-Puro in a 20:1 ratio into parental MEFs (Clontech), which
contained the transcriptional transactivator (tTa), with
Lipofectamine (Invitrogen). Cells were placed under puromycin
selection at 48 hours post transfection, and colonies were selected
after 2-4 weeks for doxycycline-dependent expression of HA-onco-Dbl
using HA.11 antibody (Covance). Cells were maintained in DMEM
supplemented with 10% (v/v) Tet system-approved FBS (clontech) and
100 .mu.g/mL G418 (Gibco). To suppress Dbl expression, 1 .mu.g/mL
doxycycline was added to the medium every 2 days. Cells were
induced by re-plating in doxycycline-free medium where residual
doxycycline was removed by replacing the medium 3 hours after
plating. Immunofluorescence was conducted on cells grown 48-72
hours on glass cover slips and fixed with 3.7% formaldehyde. Fixed
cells were permeabilized with 0.2% Triton-X100 and co-stained with
.alpha.-HA (rabbit polyclonal, Covance) and .alpha.-actin (mouse
monoclonal, Covance) for 1 hour at 37.degree. C., followed by
incubation with Oregon green-conjugated .alpha.-rabbit IgG
(Molecular Probes) and Rhodamine conjugated .alpha.-mouse IgG
(molecular probes). Digital images were collected using a Zeiss
fluorescence microscope and AxioVision 3.1 software.
Example 2--Metabolic Tracing and Foci Forming Assays
[0157] Foci forming assays were performed on Dbl-inducible cell
lines in both the induced and noninduced states by mixing with
parental MEFs (Clontech) in a 1:4 ratio. Cells were grown to
confluence in complete medium and then switched to 5% FBS and grown
for 14-21 days. The small molecule inhibitor, 968, was added to
examine its ability to inhibit focus formation. Cells were fixed in
3.7% formaldehyde and stained with 0.4% crystal violet in
methanol.
[0158] Methods for mass isotopologue distribution analyses using
[U-.sup.13C]glutamine or [U-.sup.13C]glucose (Cambridge Isotope
Laboratories) were adapted from Cheng et al., "Pyruvate Carboxylase
Is Required for Glutamine-Independent Growth of Tumor Cells," Proc.
Natl. Acad. Sci. 108:8674-79 (2011), which is hereby incorporated
by reference in its entirety. Briefly, induced (16 hours in
complete media without Dox) or non-induced cells (complete media
with 1 .mu.g/mL Dox) were plated into 100 mm.sup.2 dishes at a
density such that the cells were approximately 60% confluent when
adhered, at which point the cells were washed with PBS and grown to
80% confluence in DMEM supplemented with 1% Tet-approved FBS and 8
.mu.M 968/27 for drug treated samples, or 0.8% DMSO for control
samples, with or without 1 .mu.g/mL Dox (i.e. overnight). When 80%
confluent, cells were then washed with PBS and .sup.13C-labeling
media containing the appropriate drug, DMSO, and Dox
concentrations. Media was prepared from glucose- and glutamine-free
DMEM powder (Sigma) and supplemented with 1% Tet-approved FBS, 2 mM
L[U-.sup.13C]glutamine and 15 mM unlabeled glucose or 15 mM
D[U-.sup.13C]glucose and 2 mM unlabeled glutamine (FIGS. 2F-G),
together with 1 .mu.g/mL Dox for non-induced samples. For samples
containing [U-.sup.13C]glutamine, cells were incubated for 1 hour
before extracting metabolites, whereas samples containing
[U-.sup.13C]glucose were incubated for 8 hours. Metabolites were
extracted by first washing cells twice with ice cold normal saline
(0.9% w/v NaCl), followed by addition of 0.5 mL of a 1:1
methanol:water mixture (-20.degree. C.). An internal standard (50
nmol of 2-oxobutyrate) was added and samples were subjected to 3
freeze-thaw cycles, after which macromolecules were separated by
centrifugation. The supernatant was evaporated completely and
remaining metabolites silylated in 100 .mu.L of a trimethylsilyl
donor (Tri-Sil, Thermo) for 30 minutes at 42.degree. C. Metabolites
were subjected to GC-MS analysis using an Agilent 6970 gas
chromatograph networked to an Agilent 5973 mass selective detector.
Metabolites were identified and isotope enrichment calculated as
described by Cheng et al., "Pyruvate Carboxylase Is Required for
Glutamine-Independent Growth of Tumor Cells," Proc. Natl. Acad.
Sci. 108:8674-79 (2011), which is hereby incorporated by reference
in its entirety.
Example 3--Recombinant Glutaminase Preparation and Labeling with
Spectroscopic Probes
[0159] A plasmid encoding the mouse kidney-type glutaminase isoform
2 (GAC, GenBank Accession No. NP_001106854.1, which is hereby
incorporated by reference in its entirety) (residues 72-603) was
cloned into a pET23a vector containing an N-terminal histidine
(His)-tag and thrombin cleavage site. The expressed protein was
purified using Co.sup.2+ affinity beads (Clontech), followed by
His-tag cleavage with human thrombin (Haemetologic Technologies)
overnight at 4.degree. C., and subsequently purified by anion
exchange (GE healthcare) and gel filtration chromatography.
Purified GAC was stored in a high salt-containing buffer (20 mM
Tris-HCl pH 8.5, 500 mM NaCl, 1 mM NaN3) at -80.degree. C.,
following snap freezing in liquid N.sub.2 for long term use.
Labeling recombinant GAC with small molecule probes was performed
by exchanging 1.5 mg of enzyme into 50 mM HEPES, pH 7.2, and 100 mM
NaCl (labeling buffer), using a PD10 desalting column (GE
healthcare). The enzyme was then incubated with 50 .mu.M (5-fold
excess of enzyme) of either AlexaFluor 488 succinimidyl ester or
QSY9 succinimidyl ester (Molecular Probes) for 1 hour at 4.degree.
C. After 1 hour, the labeling reaction was quenched with 150 mM
Tris-HCl, pH 8.5, and unreacted probe was separated from
labeled-enzyme using a PD10 desalting column, eluting labeled-GAC
back into the high salt-containing buffer. The stoichiometry of
labeling was determined using the probe manufacturer's reported
molar absorptivity values and instructions. Briefly, the
concentration of GAC was determined by first correcting absorbance
at 280 nm (Abs280) for each probe, using the reported correction
values (0.11 for AlexaFluor 488 and 0.23 for QSY-9) and their
absorbance values at their absorbance maxima (495 nm and 562 nm,
respectively). The corrected Abs280 was used along with the
experimentally determined molar absorptivity of GAC
(.epsilon.280=38,850 M-1 cm-1). AlexaFluor 488 and QSY-9 probes
were quantified using their respective absorbance maxima and the
manufacturer's reported molar absorptivity values
(.epsilon.495=71,000 M-1 cm-1 and .epsilon.562=85,000 M-1 cm-1).
The labeling stoichiometries for 488-labeled and QSY-9 labeled GAC
were found to be 0.49.+-.0.09 and 1.00.+-.0.05 respectively, where
stoichiometry is defined as moles probe/moles protein.
Example 4--FRET Assays
[0160] Fluorescence experiments were performed using a Varian Cary
Eclipse Fluorometer in the counting mode. Excitation and emission
wavelengths were 490 and 520 nm, respectively. Experiments were all
performed using 1-mL samples with continuous stirring at 20.degree.
C. in 50 mM Tris-Acetate, pH 8.5, 0.1 mM ethylenediamine
tetraacetic acid (EDTA). For wild-type (WT) GAC titrations in the
absence of inorganic phosphate, 25 nM 488-GAC was equilibrated with
25 .mu.L of QSY9-GAC (at varying concentrations) and allowed to
equilibrate for 10 minutes, at which point 75 .mu.L of the
appropriate concentration of unlabeled WT GAC was added to provide
a 10-fold excess over labeled-GAC. To test whether the purified GAC
mutants can form oligomers with WT GAC, 200 nM QSY9-GAC (D391K) or
200 nM QSY9-GAC (D391K, K316E, R459E) was added to an equilibrated
sample of 20 nM 488-WT GAC. When assaying the effects of BPTES and
968 on oligomer formation, BPTES or 968 was added following
equilibration of a sample of 25 nM QSY9-WT GAC and 25 nM 488-WT
GAC. Both BPTES and 968 were prepared in DMSO, and appropriate
dilutions were made so that less than 2% (v/v) DMSO was added to an
experimental sample.
Example 5--Real-Time 968 Binding and Enzyme Activity Assays
[0161] Real-time fluorescence monitoring of 968 binding and GAC
activity through production of NADH was performed on a Varian Cary
Eclipse Fluorometer, whereas small molecule inhibition and binding
titrations were performed in a 96-well format in a Tecan Saphire
absorbance and fluorescence plate reader. Samples for monitoring
real-time binding of 968 to 488-GAC were prepared by adding 10
.mu.L of varying concentrations of 968 prepared in DMSO to an
equilibrated 1 mL sample of 10 nM 488-GAC, while observing 488
fluorescence (490 nm excitation/520 nm emission). Similarly, this
method was replicated for monitoring real-time binding of 968 to
mutant forms of GAC, namely 488-labeled GAC (GD391K) and 488-GAC
(D391K, K316E, R459E). Real-time activity assays monitoring 968
binding and NADH production were prepared in 1 mL samples, where 10
units of glutamate dehydrogenase (Sigma) and 2 mM NAD+(Sigma) were
prepared in 50 mM Tris-Acetate, pH 8.5, 0.1 mM EDTA and
equilibrated at 20.degree. C. WT GAC (10 nM) was added and allowed
to equilibrate 2 minutes before monitoring the fluorescence
emission of 488-GAC (490 nm excitation, 520 nm emission) and NADH
(340 nm excitation, 490 nm emission). Appropriate dilutions of 968
or BPTES prepared in DMSO were introduced after 30 seconds and
allowed to equilibrate for 2 minutes before a 180 .mu.L solution of
K.sub.2HPO.sub.4 and glutamine was added to make a final
concentration of 50 mM K.sub.2HPO.sub.4 and 20 mM glutamine, to
initiate GAC activation. The activity of GAC was measured in a
coupled assay, by monitoring the NADH produced by glutamate
dehydrogenase, which converts the product of the
glutaminase-catalyzed reaction, glutamate, to .alpha.-ketoglutarate
and ammonia by reducing NAD+ to NADH. Because solutions containing
glutamine undergo non-enzymatic degradation to glutamate, samples
were further analyzed by subtracting the NADH produced by
glutaminase in the presence of 968, BPTES, or the equivalent volume
of DMSO as a control, from the NADH produced in the absence of
glutaminase under identical experimental conditions. NADH was
quantified using a standard curve of freshly prepared NADH (Sigma)
in 50 mM Tris-Acetate, pH 8.5, and 0.1 mM EDTA.
[0162] Procedures for the real-time binding and inhibition assays
were adapted for 96-well microtiter format with minor alterations.
Briefly, 2 .mu.L of inhibitor or DMSO were distributed across the
96-well plate, followed by addition of 200 .mu.L of 10 nM 488-GAC,
unlabeled WTGAC, or in the absence of added GAC as a negative
control, in 50 mM Tris-Acetate, pH 8.5, and 0.1 mM EDTA, followed
by immediate monitoring of 488 fluorescence (490 nm/520 nm
excitation/emission, 5 nm/20 nm excitation/emission slits). The
488-fluorescence was measured every 2 minutes with 90 seconds of
orbital shaking, followed by 30 seconds resting between each cycle
for a total of four cycles (i.e. 6 minutes). A mixture of GDH and
NAD+(20 .mu.L) was then added to give 10 units of GDH and 2 mM
NAD+. To activate GAC, 30 .mu.L of a mixture of glutamine and
K.sub.2HPO.sub.4 were added to give a total concentration of 50 mM
K.sub.2HPO.sub.4 and 20 mM glutamine in each well. NADH
fluorescence was measured (340 nm/460 nm excitation/emission, 10
nm/10 nm excitation/emission slits) every minute with 30 seconds
orbital shaking, and a 30 second rest between each reading, for 10
cycles (i.e. 9 minutes). Three wells were prepared for each
experimental condition (i.e. each concentration of compound)
alongside one well where 2 .mu.L of DMSO were added in place of
inhibitor, and one well that contained the small molecule inhibitor
but no GAC. To analyze 488-quenching by the added compound,
488-fluorescence (F) was normalized to the DMSO control (F0).
Quenching was quantified as follows: 1-F/F0. For compounds that
emitted fluorescence within the observed range, fluorescence
measured in the well that contained the compound but lacked GAC was
used to subtract added fluorescence due to the compound. Similarly,
samples were analyzed for NADH fluorescence by subtracting the
fluorescence measured for the experimental condition from the NADH
fluorescence in the well that contained the added compound but no
GAC. Percent inhibition at each drug concentration was calculated
using the adjacent DMSO control.
Example 6--End-Point Glutaminase Activity Assays
[0163] Activity assays used to compare FRET values and evaluate the
activity of GAC mutants followed a two-step protocol adapted from
Robinson et al., "Novel Mechanism of Inhibition of Rat Kidney-Type
Glutaminsae by Bis-2-(5-Phenylacetamido-1,2,4-Thiadiazol-2-yl)Ethyl
Sulfide (BPTES)," Biochem. J. 406(3):407-14 (2007), which is hereby
incorporated by reference in its entirety). Briefly, 20 .mu.L of 20
mM glutamine, 50 mM Tris-acetate, pH 8.5, and 0.1 mM EDTA, in
either the presence or absence of a K.sub.2HPO.sub.4, were added to
a UV-transparent Costar 96-well plate (Corning). To initiate the
reaction, 5 .mu.L of a solution of the appropriate concentration of
GAC, prepared in 20 mM Tris-HCl, pH 8.5, 100 mM NaCl, and 1 mM
NaN.sub.3, were added to the glutamine solution and incubated at
23.degree. C. for 2 minutes before the reaction was quenched using
2.5 .mu.L of 3 M HCl. For reactions that contained more than 250 nM
GAC, the first reaction was quenched at 30 seconds instead of 2
minutes. The second step was initiated by the addition of 200 .mu.L
of 12 units/.mu.L GDH, 2 mM NAD+, 100 mM hydrazine (Sigma), and 100
mM Tris-HCl, pH 9.2, on top of the first quenched reaction and
incubated for 45 minutes at 23.degree. C. before reading NADH
absorbance. Glutamate produced by the first reaction was determined
from the amount of NADH generated in the second reaction by using
the extinction coefficient for NADH (6,220 M-1 cm-1).
Example 7--Multi-Angle Light Scattering (MALS)
[0164] Purified GAC and GAC mutants were subjected to multi-angle
light scattering (MALS) as previously described by Moller et al.,
"Small Angle X-Ray Scattering Studies of Mitochondrial Glutaminase
C Reveal Extended Flexible Regions, and Link Oligomeric State with
Enzyme Activity," PLoS One 8(9):e74783 (2013), which is hereby
incorporated by reference in its entirety. Briefly, 50 .mu.L
samples of 5 mg/mL GAC were injected onto a WTC-030S5
size-exclusion column (Wyatt technology), coupled to a static
18-angle light scattering detector (DAWN HELEOS-II) and a
refractive index detector (OptiLab TrEX, Wyatt Technology), at
23.degree. C. The size-exclusion column was equilibrated with 20 mM
Tris-HCl, pH 8.5, and 200 mM NaCl with or without 50 mM
K.sub.2HPO.sub.4. The flow rate was kept at 1 mL/minutes. RMS
radius and mass distribution (polydispersity) were analyzed using
the ASTRA software, with monomeric BSA (Sigma) serving to normalize
the light scattering signal.
Example 8--Preparation of 968 Analogs
[0165] Compounds 27 (see FIG. 5D(i)) and 742 (see FIG. 5D(g)) were
obtained from Chembridge (San Diego), and compound 031 was from
Specs (Netherlands). Starting materials for chemical synthesis were
used without further purification.
Example 9--3-Bromo-4-(Dimethylamino)Benzaldehyde (4)
[0166] 4-(Dimethylamino)benzaldehyde (6.7 mmol) was suspended in
1,4 dioxane (13 mL), Nbromosuccinimide (7.0 mmol) was added at once
and stirred at room temperature for 3 hours. Water (50 mL) and
ethyl acetate (50 mL) were added and the organic layer washed with
two more portions of water (50 mL) then removed and dried with
magnesium sulfate. The solvent was removed by rotary evaporation
and the crude product purified by silica gel chromatography using
2:1 CH.sub.2Cl.sub.2:hexane as the eluent to give 0.84 g of a clear
oil (55% yield). 1H NMR (400 MHz, CDCl3): .delta.=9.80 (1H, s),
8.03 (1H, d, J=1.84 Hz), 7.73 (1H, dd, J=8.24 Hz, 1.84 Hz), 7.06
(1H, d, J=8.24 Hz), 2.94 (6H, s).
Example 10--General Procedure for Synthesis of 968, SU-1, and SU-14
(5)
[0167] The appropriate arylamine,
3-bromo-4-(dimethylamino)benzaldehyde and dimedone (2 mmol each)
were heated to reflux in 10 mL ethanol for 1 hour, upon which time
a precipitate formed. The precipitate was filtered, washed with
ethanol and dried to give the product.
[0168] 968: .sup.1H NMR (400 MHz, d6-DMSO): .delta.=9.71 (1H, s),
7.91 (1H, d, J=8.7 Hz), 7.76 (2H, m), 7.40 (1H, m), 7.35 (1H, m),
7.28 (2H, m), 7.10 (1H, dd, J=8.24 Hz, 1.8 Hz), 6.90 (1H, d, J=8.24
Hz), 5.70 (1H, s), 2.52 (6H, s), 2.47 (1H, d, J=15.9 Hz) 2.37 (1H,
d, J=15.9 Hz), 2.18 (1H, d, J=16.0 Hz), 2.02 (1H, d, J=16.0 Hz),
0.99 (3H, s), 0.85 (3H, s).
[0169] SU-1: .sup.1H NMR (400 MHz, d6-DMSO): .delta.=9.28 (1H, s),
8.40 (1H, d, J=8.9 Hz), 7.78 (1H, d, J=8.9 Hz), 7.55 (1H, m), 7.45
(2H, m), 7.36 (1H, m), 7.25 (1H, d, J=8.1 Hz), 7.11 (1H, m), 6.94
(1H, d, J=8.8 Hz), 5.11 (1H, s), 2.73 (1H, d, J=15.1 Hz), 2.64 (1H,
d, J=15.1 Hz), 2.55 (6H, s), 2.22 (1H, d, J=16.0 Hz), 2.04 (1H, d,
J=16.0 Hz), 1.02 (3H, s), 0.97 (3H, s). SU-14: .sup.1H NMR
(d6-DMSO): .delta.=9.77 (1H, s), 8.56 (1H, s), 8.43 (1H, s), 8.07
(1H, d, J=7.8 Hz), 7.94 (2H, d, J=9.16 Hz), 7.49 (1H, m), 7.40 (2H,
m), 7.31 (1H, m), 7.26 (1H, m), 6.90 (1H, d, J=8.3 Hz), 5.88 (1H,
s), 2.55 (1H, d, J=16.0 Hz), 2.50 (6H, s) 2.42 (1H, d, J=16.0 Hz),
2.22 (1H, d, J=16.0 Hz), 2.05 (1H, d, J=16.0 Hz), 1.02 (3H, s),
0.88 (3H, s).
Example 11--General Procedure for Synthesis of SU-2, SU-7, SU-8
[0170] The appropriate arylamine (3.7 mmol) and
3-bromo-4-(dimethylamino)benzaldehyde (3.7 mmol) were dissolved in
benzene (5 mL) supplemented with molecular sieves (0.7 g) and
stirred at RT for 18 hours. The mixture was filtered and the solid
material washed with dichloromethane. The combined filtrate was
dried with magnesium sulfate, filtered and the solvent removed by
rotary evaporation. The crude imine was dissolved in 1-butanol (6
mL), combined with dimedone (2 mmol) and heated to reflux for 2
hours. The reaction mixture was cooled and solvent removed by
rotary evaporation. The crude product was purified by silica gel
chromatography using 1:1 hexanes:ethyl acetate as the eluent.
[0171] SU-2: .sup.1H NMR (400 MHz, CDCl3): .delta.=7.35 (1H, d,
J=1.7 Hz), 7.1-7.25 (3H, m), 6.96 (1H, dd J=1.7 Hz, 11 Hz), 6.92
(1H, d, J=11 Hz), 6.75 (1H, d, J=8.9 Hz), 6.61 (1H, br s), 5.19
(1H, s), 2.69 (6H, s), 2.38 (1H, d, J=8.0 Hz), 2.34 (2H, m), 2.24
(1H, d, J=8 Hz), 1.09 (3H, s), 1.12 (3H, s).
[0172] SU-7: .sup.1H NMR (400 MHz, CDCl3): .delta.=7.30 (1H, d,
J=1.8 Hz), 7.18 (1H, dd, J=8.2 Hz, 1.8 Hz), 7.06 (2H, m), 6.93 (1H,
d, J=8.3 Hz), 6.69 (1H, d, J=9.1 Hz), 6.20 (1H, br s), 5.11 (1H,
s), 2.74 (2H, m), 2.72 (6H, s), 2.40 (1H, d, J=11.4 Hz), 2.22 (1H,
d, J=9.1 Hz), 1.09 (3H, s), 1.03 (3H, s).
[0173] SU-8: .sup.1H NMR (400 MHz, d6-DMSO): .delta.=7.33 (1H, d,
J=1.7 Hz), 7.1-7.2 (3H, m), 6.92 (1H, d, J=11 Hz), 6.70 (1H, d,
J=11 Hz), 6.34 (1H, br s), 5.19 (1H, s), 2.70 (6H, s), 2.38 (2H,
m), 2.22 (2H, m) 1.23 (9H, s), 1.09 (3H, s), 1.02 (3H, s).
Example 12--The GAC Inhibitor 968 Blocks Glutaminolysis in
Transformed Cells
[0174] Previous work identified a potential connection between
glutamine metabolism and Rho GTPase-dependent oncogenic
transformation, through the discovery of the small molecule
inhibitor 968 (Wang et al., "Targeting Mitochondrial Glutaminase
Activity Inhibits Oncogenic Transformation," Cancer Cell
18(3):207-19 (2010), which is hereby incorporated by reference in
its entirety). It was shown that 968 specifically inhibited the
growth of transformed cells and various cancer cells by blocking
GAC activation, although the detailed mechanism was unclear. Here,
it has been set out to better understand how 968 functions by first
examining its effects on glutamine metabolism in a well-defined
model system for oncogenic transformation, in which the stable
expression of the Dbl oncogene in mouse embryonic fibroblasts
(MEFs) is controlled by the removal of doxycycline (Dox).
[0175] As shown in FIG. 1A, induction of oncogenic Dbl in MEFs
results in marked changes in cell morphology, as a result of
cytoskeletal rearrangements caused by the activation of Rho GTPases
(Lin et al., "Specific Contributions of the Small GTPases Rho, Rac,
and Cdc42 to Dbl Transformation," J. Biol. Chem. 274(33):23633-41
(1999); Rossman et al., "GEF Means Go: Turning on Rho GTPases with
Guanine-Nucleotide Exchange Factors," Nat. Rev. Mol. Cell. Biol.
6(2):167-80 (2005); Etienne-Manneville and Hall, "Rho GTPases in
Cell Biology," Nature 420(6916):629-35 (2002); Olivo et al.,
"Distinct Involvement of Cdc-42 and RhoA GTPases in Actin
Organization and Cell Shape in Untransformed and Dbl Oncogene
Transformed NIH3T3 Cells," Oncogene 19(11):1428-36 (2000), which
are hereby incorporated by reference in their entirety). These
morphological changes accompany the ability of oncogenic
Dbl-expressing cells to overcome contact inhibition to form foci.
Consistent with previous results (Wang et al., "Targeting
Mitochondrial Glutaminase Activity Inhibits Oncogenic
Transformation," Cancer Cell 18(3):207-19 (2010), which is hereby
incorporated by reference in its entirety), treatment of MEFs
expressing oncogenic Dbl with 968 blocked focus formation (FIG.
1B). It was then examined whether these effects were accompanied by
an inhibition of glutaminolysis. As shown in FIGS. 1C and 1D, the
induction of oncogenic Dbl expression in MEFs increased
glutaminolysis and glutamine dependent anaplerosis, as monitored by
.sup.13C enrichment in TCA cycle intermediates derived from
[U-.sup.13C]-glutamine. As shown in FIGS. 2A-F, treatment of
Dbl-expressing cells with 968 caused significant reductions in the
glutamine-derived .sup.13C isotopic enrichment within TCA cycle
intermediates, but did not result in the depletion of relative pool
sizes of each metabolite, with the exception of a modest reduction
in intracellular glutamate (compare red (b) and yellow (d) versus
black (a), green (b), and blue (e)). A modest inhibition of
glutaminolysis by 968 was also observed in MEFs not expressing Dbl
(see FIG. 2B and the M+5 histograms for Dbl-OFF, .+-.968, in FIG.
2C, and the M+4 histograms in FIGS. 2D-F). However, these effects
were not accompanied by reductions in cell growth, suggesting that
glutamine metabolism is critical for supporting the transformed
phenotypes accompanying oncogenic Dbl expression, but not for the
proliferative capability of normal MEFs. Treatment with the less
potent 968-analogue, compound 27, caused a weaker inhibition of the
.sup.13C enrichment of metabolites (see FIG. 1D), consistent with
its reduced potency to inhibit enzymatic activity (see below).
[0176] As shown in FIG. 2G, oncogenic Dbl induction did not cause
marked increases in glucose-fueled anaplerosis, as measured by
.sup.DC enrichment in citrate, when using [U-.sup.13C]-glucose as a
tracer (see the M+2 histograms in FIG. 2G), demonstrating that a
highly specific stimulation of glutamine metabolism accompanies Rho
GTPase-dependent transformation. However, 968 was observed to
inhibit glucose labeling of citrate isotopologues (see the M+2
histogram in FIG. 2H). This presumably was due to the inhibition of
glutamate flux by 968. Overall, these results show how 968
attenuates cellular glutamine metabolism and restores a normal
growth phenotype in cells expressing oncogenic Dbl, thus
highlighting the role of glutamine as a critical source for
anaplerosis during cellular transformation.
Example 13--Examining the Effects of 968 on the Dimer-to-Tetramer
Transition of GAC
[0177] The transition of GAC from a dimer to a tetramer has been
suggested to be essential for enzyme activity (Robinson et al.,
"Novel Mechanism of Inhibition of Rat Kidney-Type Glutaminsae by
Bis-2-(5-Phenylacetamido-1,2,4-Thiadiazol-2-yl)Ethyl Sulfide
(BPTES)," Biochem. J. 406(3):407-14 (2007); Godfrey et al.,
"Correlation Between Activation and Dimer Formation of Rat Renal
Phosphate-Dependent Glutaminase," J. Biol. Chem. 252(6):1927-31
(1977); Kenny et al., "Bacterial Expression, Purification, and
Characterization of Rat Kidney-Type Mitochondrial Glutaminase,"
Protein Expr. Purif. 31(1):140-48 (2003), which are hereby
incorporated by reference in their entirety). A well-established
allosteric inhibitor of GAC, BPTES, has been shown to stabilize an
inactive tetrameric state of the enzyme (Robinson et al., "Novel
Mechanism of Inhibition of Rat Kidney-Type Glutaminsae by
Bis-2-(5-Phenylacetamido-1,2,4-Thiadiazol-2-yl)Ethyl Sulfide
(BPTES)," Biochem. J. 406(3):407-14 (2007), which is hereby
incorporated by reference in its entirety). Thus, it was examined
whether 968 acted in a similar manner, by developing a real-time
read-out for the GAC dimer-to-tetramer transition. FIG. 3A depicts
the proposed FRET assay, where oligomer formation is monitored
between two populations of purified recombinant GAC, labeled with
either the highly fluorescent AlexaFluor 488 (donor) probe, or with
the non-fluorescent QSY9 (acceptor) chromophore. A major advantage
of using FRET as a direct read-out for GAC tetramer formation is
the ability to monitor oligomer formation at the low concentrations
of GAC commonly used for assaying its enzymatic activity.
[0178] The addition of QSY9-GAC to 488-GAC yielded a dose-dependent
quenching of the donor 488 emission due to FRET, that was
reversible upon the addition of unlabeled GAC, demonstrating that
GAC tetramer formation is a dynamic process (see FIG. 3B). The dose
dependent binding isotherm obtained from the QSY9-GAC titration
profile directly correlated with the basal activation of GAC that
occurs at increasing protein concentrations (i.e. due to tetramer
formation through mass-action), yielding an apparent K.sub.D of 164
nM (.+-.20 nM) for tetramer formation (see FIG. 3C), supporting the
contention that the GAC tetramer is the minimal unit for enzymatic
activity.
[0179] The effects of 968 were then compared, versus BPTES, on the
GAC dimer-to-tetramer transition. Consistent with previous findings
that BPTES stabilizes GAC as an inactive tetramer (DeLaBarre et
al., "Full-Length Human Glutaminase in Complex with an Allosteric
Inhibitor," Biochemistry 50(50):10764-70 (2011), which is hereby
incorporated by reference in its entirety), it was found that it
caused an immediate quenching of 488-GAC fluorescence emission when
added to a mixture of 488-GAC and QSY9-GAC (see FIG. 3D), i.e. due
to the ability of BPTES to promote the formation of
488-GAC:QSY9-GAC (donor:acceptor) tetramers. These stable GAC:BPTES
tetrameric complexes were less susceptible to reversal by the
addition of unlabeled GAC (in FIG. 3D, compare the "a" trace for
the addition of unlabeled GAC in the presence of the vehicle
control DMSO to the "e" trace, which represents the addition of
unlabeled GAC in the presence of 5 .mu.M BPTES). Interestingly, 968
elicited a markedly different response, causing a significant
change in the fluorescence emission of 488-GAC, followed by a
partial fluorescence recovery upon the addition of excess unlabeled
GAC (see FIG. 3E). The recovery of 488 fluorescence when adding
excess unlabeled GAC was due to the elimination of FRET between
488-GAC and QSY9-GAC, following the formation of mixed tetramers
between 488-GAC or QSY9-GAC and unlabeled GAC. Thus, 968 does not
appear to interfere with GAC tetramer formation. However, the
inability to achieve a full recovery of the fluorescence emission
suggested that 968 binding was directly affecting 488-GAC donor
fluorescence emission. Indeed, it was found that 968 caused a
dose-dependent quenching of 488-GAC emission (in the absence of the
FRET acceptor QSY9-GAC) that matched the 968-mediated inhibition of
GAC activity (see FIG. 3F and FIG. 4). Taken together, these
findings show that 968 does not mimic the actions of BPTES by
trapping GAC in an inactive tetrameric state, but instead regulates
GAC activity through a distinct allosteric mechanism.
Example 14--Real-Time Monitoring of 968 Binding to GAC and its
Inhibition of Enzyme Activity
[0180] A real-time enzyme activity assay was developed so as to
simultaneously monitor both the binding of 968 to GAC and its
effects on enzyme activity. The enzymatic activity of 488-GAC is
assayed by monitoring NADH production (i.e. fluorescence emission
at 460 nm) that accompanies the conversion of glutamate (the
product of the GAC-catalyzed reaction) to .alpha.-ketoglutarate,
catalyzed by glutamate dehydrogenase. FIG. 5A depicts the coupling
of these two fluorescence assays, and FIG. 5B shows the results of
an experiment simultaneously monitoring the direct binding of 968
to GAC ("a", solid line) and its inhibition of enzyme activity
("b", solid line; the dashed line represents the control enzyme
activity treated with the solvent vehicle DMSO). Unlike 968, BPTES
does not directly affect 488-GAC fluorescence, under conditions
where it strongly inhibits GAC activity (in FIG. 5B, see the "a"
and "b" dotted traces, respectively).
[0181] These assays were then adapted to a 96-well plate format,
and it was shown that 968 exhibited an overlapping dose-dependent
inhibition of both 488-GAC and unlabeled GAC activity (FIG. 5C,
black closed and open circles respectively), as well as an
overlapping dose-response for its direct binding to 488-GAC (FIG.
5C, grey closed circles). The robustness of these high throughput
binding and enzymatic assays was tested by examining a group of
newly synthesized 968-derivatives (compounds SU-1, SU-2, SU-7,
SU-8, and SU-14 in FIG. 5D), together with molecules 031,27, and
742 (see FIG. 5D) that were previously characterized and shown to
be GAC inhibitors (Wang et al., "Targeting Mitochondrial
Glutaminase Activity Inhibits Oncogenic Transformation," Cancer
Cell 18(3):207-19 (2010); Katt et al., "Dibenzophenanthridines as
Inhibitors of Glutaminase C and Cancer Cell Proliferation," Mol.
Cancer Ther. 11(6):1269-78 (2012), which are hereby incorporated by
reference in their entirety). A direct correlation exists between
the ability of different 968 analogs to bind to GAC and to inhibit
its enzymatic activity (see FIG. 5E). The results of these
analyses, and particularly the finding that substituting the
napthyl group of 968 with a quinoline moiety (e.g. compound 27)
markedly affected both binding and inhibitory activity, suggests
that hydrophobicity at this position is required for maximal
efficacy.
[0182] Previous studies of the 968-mediated inhibition of
recombinant GAC activity showed that 968 was much more effective
when it was added prior to glutamine and inorganic phosphate (the
latter being an allosteric activator that stimulates GAC tetramer
formation and GAC activity), compared to when it was added after
the addition of phosphate (Katt et al., "Dibenzophenanthridines as
Inhibitors of Glutaminase C and Cancer Cell Proliferation," Mol.
Cancer Ther. 11(6):1269-78 (2012), which is hereby incorporated by
reference in its entirety). Therefore, whether the ability of 968
to bind to GAC was compromised under conditions where the enzyme
was pre-treated with inorganic phosphate and assumed an activated
tetrameric state was examined. In fact, it was found that 968 was
capable of binding to a tetrameric GAC species comprised of
488-labeled GAC and QSY9-labeled GAC dimers, as read-out by the
quenching of 488 fluorescence emission (FIGS. 6A and 6B). Moreover,
968 was able to bind to GAC that had been preincubated with
inorganic phosphate (FIG. 6C, closed versus open circles). However,
under these conditions, 968 was much less effective at inhibiting
enzyme activity (FIG. 6D, closed versus open circles). Thus,
phosphate induces an activated state that is less sensitive to 968
inhibition, even though 968 is able to bind to phosphate-activated
GAC. In contrast, when GAC was pre-incubated with 968 before adding
phosphate, the enzyme activity was strongly inhibited and directly
correlated with the binding of 968 (FIGS. 6C and 6D, open
circles).
Example 15--968 Preferentially Binds to the Monomeric State of
GAC
[0183] Docking analyses using the x-ray structure of the GAC
tetramer, together with mutagenesis studies, suggested that 968
binds in a cove between the monomer-monomer interface (Katt et al.,
"Dibenzophenanthridines as Inhibitors of Glutaminase C and Cancer
Cell Proliferation," Mol. Cancer Ther. 11(6):1269-78 (2012), which
is hereby incorporated by reference in its entirety). To examine
the ability of 968 to bind to different oligomeric states of GAC,
the recently solved x-ray structures of GAC (DeLaBarre et al.,
"Full-Length Human Glutaminase in Complex with an Allosteric
Inhibitor," Biochemistry 50(50):10764-70, which is hereby
incorporated by reference in its entirety) was used to design
mutants trapped as either monomers or dimers. FIG. 7A depicts the
BPTES-binding sites within the GAC tetramer interface and the
proposed 968-binding pocket at the C-terminal region of the
monomer-monomer interface. Residue contacts that were mutated in
order to create constitutive monomeric and dimeric GAC mutants are
highlighted at the GAC-tetramer helical interface (bottom inset),
as well as at the GAC dimer interface (top inset). When a point
mutation was incorporated at the tetramer interface of mouse GAC
(D391K), tetramer formation was disrupted with the resulting GAC
mutant being trapped in a dimeric state, as determined by
multi-angle light scattering (MALS) (FIG. 7B, "b" trace) and
further confirmed with the determination of its x-ray crystal
structure (PDB submission in preparation). Introducing point
mutations at the dimer interface of mouse GAC (K316E, R459E),
within the background of the dimeric GAC mutant, resulted in a
monomeric GAC (D391K, K316E, R459E) species (FIG. 7B, "c" trace).
As expected, the monomeric and dimeric GAC mutants showed neither a
concentration-dependent basal enzymatic activity, nor
phosphate-stimulated activity (FIGS. 7C-D). While the addition of
wildtype (WT) QSY9-labeled GAC to WT 488-labeled GAC resulted in
the expected FRET due to tetramer formation (FIG. 7E, "a" trace),
the addition of either the QSY9-labeled GAC (D391K) dimer or the
GAC (D391K, K316E, R459E) monomer to WT 488-labeled GAC failed to
result in a significant quenching of the 488-donor fluorescence
(FIG. 7E, "b" and "c" traces, respectively). The addition of the
dimeric QSY9-GAC (D391K) to WT 488-GAC did induce a minor quenching
of the 488-GAC emission, however, this was most likely due to the
formation of mixed donor and acceptor labeled dimers, which result
from a relatively minor exchange of the monomeric GAC units.
[0184] It was found that 968 was capable of binding to WT 488-GAC,
as well as to both the dimeric GAC (D391K) and the monomeric GAC
(D391K, K316E, R459E), with the monomeric GAC having the highest
affinity for 968 (FIG. 7F). These results suggested that 968 should
be most effective at inhibiting WT GAC at relatively low enzyme
concentrations, i.e. where equilibrium conditions favor GAC
initially existing as a monomer. FIG. 7G shows that when the
concentration of GAC was decreased from 50 nM to 5 nM, 968 was able
to inhibit GAC activity with greater potency. Furthermore, the
968-mediated inhibition of GAC activity at these low enzyme
concentrations correlated well with its inhibition of oncogenic
transformation (FIG. 7G).
Discussion of Examples 1-15
[0185] Previous work aimed at identifying inhibitors that
specifically block Rho GTPase-dependent transformation led to the
discovery of the benzophenanthridinone 968 (Wang et al., "Targeting
Mitochondrial Glutaminase Activity Inhibits Oncogenic
Transformation," Cancer Cell 18(3):207-19 (2010), which is hereby
incorporated by reference in its entirety). Unexpectedly, the
protein target for 968 appeared to be a specific splice variant
(GAC) of a family of enzymes collectively called glutaminase, that
catalyzes the hydrolysis of glutamine to glutamate with the
production of ammonia. This highlighted a previously unappreciated
connection between the roles of Rho GTPases in driving oncogenic
transformation and the regulation of glutamine metabolism. Given
the striking specificity that 968 exhibited in its ability to
inhibit transformed cells and cancer cells, with little or no
effect on their normal cellular counterparts, it was of interest to
better understand how 968 functions.
[0186] An inducible expression system for oncogenic Dbl allowed the
temporal control of the expression of this upstream activator of
Rho GTPases in a well-defined manner. Using this system, a direct
correlation between the ability of 968 to prevent a key outcome of
Dbl-induced transformation, namely focus formation, and to
specifically inhibit glutaminolysis was established. Thus, the
inhibitory actions of 968 upon oncogenic transformation appear to
be a direct outcome of its ability to interfere with glutamine
metabolism.
[0187] It was then set out to understand how 968 inhibits the
activity of a key enzyme in glutamine metabolism, GAC. Because
BPTES, a well characterized allosteric inhibitor of GAC, has been
shown to bind and stabilize an inactive tetrameric form of the
enzyme, one possibility was that 968 had a similar effect. Previous
studies used analytical ultracentrifugation, gel filtration, and
electron microscopy to investigate the oligomeric transitions of
GAC; however, these analyses were performed at GAC concentrations
above the K.sub.D for tetramer formation reported here (Robinson et
al., "Novel Mechanism of Inhibition of Rat Kidney-Type Glutaminase
by Bis-2-(5-Phenylacetamido-1,2,4-Thiadiazol-2-yl)Ethyl Sulfide
(BPTES)," Biochem. J. 406(3):407-14 (2007); Godfrey et al.,
"Correlation Between Activation and Dimer Formation of Rat Renal
Phosphate-Dependent Glutaminase," J. Biol. Chem. 252(6):1927-31
(1977); Kenny et al., "Bacterial Expression, Purification, and
Characterization of Rat Kidney-Type Mitochondrial Glutaminase,"
Protein Expr. Purif. 31(1):140-48 (2003); Ferreira et al., "Active
Glutaminase C Self-Assembles into a Supratetrameric Oligomer That
Can Be Disrupted By an Allosteric Inhibitor," J. Biol. Chem.
288(39):28009-20 (2013); Moller et al., "Small Angle X-Ray
Scattering Studies of Mitochondrial Glutaminase C Reveal Extended
Flexible Regions, and Link Oligomeric State With Enzyme Activity,"
PLoS One 8(9):e74783 (2013), which are hereby incorporated by
reference in their entirety). Thus, a real-time FRET assay was used
for monitoring GAC tetramer formation. The highly sensitive FRET
assay enabled the direct monitoring of GAC tetramer formation and
showed that it correlates with enzyme activation, as well as to
compare the effects of 968 and BPTES on the dimer-to-tetramer
transition. It was found that unlike BPTES, 968 does not stabilize
an inactive tetrameric state of GAC. However, during the course of
these FRET experiments, it was discovered that the binding of 968
to GAC resulted in a quenching of the reporter group fluorescence,
thus providing a direct spectroscopic read-out for the ability of
this inhibitor and various analogs to bind to the enzyme.
[0188] By taking advantage of a direct binding assay for 968,
together with the recent development of GAC mutants that exist as
monomers or dimers, it was discovered that 968 has a marked
preference for binding to the monomeric form of the enzyme. While
968 is able to bind, albeit more weakly, to a GAC dimer, as well as
to a GAC tetramer that has been activated by the allosteric
regulator inorganic phosphate, it is unable to inhibit the activity
of the activated enzyme tetramer. Therefore, 968 preferentially
binds to an inactive, monomeric state of GAC and prevents it from
undergoing activating conformational changes, whereas, if GAC
reaches an activated state prior to 968-binding, then 968 is unable
to inhibit enzyme activity.
[0189] These findings highlight the distinction between the two
classes of allosteric GAC inhibitors for which BPTES and 968 are
the prototypes. BPTES is able to bind and inhibit activated GAC,
whereas 968 binds preferentially to and stabilizes an inactive
state of the enzyme. In addition, these results shed light on the
reason for previous discrepancies when comparing the 968
dose-dependencies for the inhibition of recombinant GAC activity
versus oncogenic transformation (Katt et al.,
"Dibenzophenanthridines as Inhibitors of Glutaminase C and Cancer
Cell Proliferation," Mol. Cancer Ther. 11(6):1269-78 (2012), which
is hereby incorporated by reference in its entirety). Specifically,
in those earlier experiments, the concentrations of recombinant GAC
routinely being assayed represented a mixture of dimers and
tetramers. Consequently, the IC.sub.50 values for 968 reflected its
weaker binding to these oligomeric GAC species. Indeed, when the
binding of 968 to GAC, together with its ability to inhibit enzyme
activity, is assayed at GAC concentrations where it initially
exists predominantly as a monomer, the dose response profiles for
these binding assays match the dose-dependent inhibition of
transformation in cell culture.
[0190] In conclusion, it was shown that 968 functions as a highly
specific inhibitor of oncogenic transformation by blocking a key
step in glutamine metabolism necessary for sustaining the
transformed state. It is demonstrated that 968 is capable of
directly binding to GAC, a key enzyme responsible for elevated
glutamine metabolism in transformed cells and cancer cells, and
that 968 preferentially binds to a monomeric, inactive state of the
enzyme. While an x-ray crystal structure of 968 bound to GAC has
not yet been achieved, these findings shed new light on why this
has been so challenging, given that crystallization trials have
been routinely performed at GAC concentrations where it exists as a
tetramer, i.e. the least favorable species for binding 968 (Brown
et al., "Functional and Structural Characterization of Four
Glutaminases From Escherichia coli and Bacillus subtilis,"
Biochemistry 47(21):5724-35 (2008); DeLaBarre et al., "Full-Length
Human Glutaminase in Complex with an Allosteric Inhibitor,"
Biochemistry 50(50):10764-70 (2011); Thangavelu et al., "Structural
Basis for the Allosteric Inhibitory Mechanism of Human Kidney-Type
Glutaminase (KGA) and Its Regulation by Raf-Mek-Erk Signaling in
Cancer Cell Metabolism," Proc. Natl. Acad. Sci. 109(20):7705-10
(2012); Cassago et al., "Mitochondrial Localization and
Structure-Based Phosphate Activation Mechanism of Glutaminase C
with Implications for Cancer Metabolism," Proc. Natl. Acad. Sci.
USA 109(4):1092-97, which are hereby incorporated by reference in
their entirety). The ability to generate monomeric GAC mutants now
provides new opportunities for achieving such a structure.
Moreover, the availability of a direct binding read-out adapted for
plate-reader assays offers exciting possibilities for the
identification of 968-like allosteric inhibitors that could yield
new therapeutic strategies against cancer.
Example 16--Synthesis of Additional Compounds
[0191] Additional compounds having a compound 968-like scaffold
were synthesized as shown below.
[0192] General Synthetic Scheme for Inhibitor Analogs of the 968
Scaffold
##STR00020##
[0193] The appropriate aryl amine, 1,3 cyclohexanedione and aryl
aldehyde were mixed in equal molar ratios and heated to reflux in
ethanol. The resulting product generally precipitates from solution
and is recovered by filtration. Representative NMRs are shown in
FIGS. 8A-B.
[0194] SU-11 and compounds having a compound SU-11-like scaffold
were synthesized as shown below.
[0195] General Synthetic Scheme for Inhibitor Analogs of the SU-11
Scaffold
##STR00021##
[0196] The appropriate aryl amine and aryl aldehyde were mixed in
equal molar amounts in dichloromethane, and 1.3 molar equivalents
of sodium triacetoxyborohydride was added and the mixture stirred
at room temperature overnight. The product was purified by silica
gel chromatography. Representative NMRs are shown in FIG. 8C.
Example 17--Real-Time Binding and Enzyme Activity Assays
[0197] Compounds SU-3, SU-6, and SU-9
[0198] Small molecule inhibition and binding titrations with
compounds SU-3, SU-6, and SU-9 (see FIG. 9 for structures) were
performed by the following procedure. The GAC inhibitors were
solvated in DMSO. Assay vessels were charged with 1 .mu.L of
inhibitor in order to effect the reported final concentration. To
each vessel, 95 .mu.L of an aqueous solution containing 48 mM
Tris-acetate (pH 8.6), 21 mM glutamine, and 50 nM recombinant GAC
was added. Fifteen .mu.L of either water or 1 M potassium
phosphate, pH 8.2, were added to the mixture to begin the reaction.
The assay reagents were incubated for 10 minutes at room
temperature, at which point 10 .mu.L of ice-cold 2.4 M hydrochloric
acid was added to quench the enzymatic reaction. A second reaction
vessel contained 218 .mu.L of an aqueous solution containing 114 mM
Tris-HCl (pH 9.4), 0.35 mM ADP, 1.7 mM b-NAD.sup.+, 238 mM
hydrazine, and 1.3 units of glutamate dehydrogenase. A third
reaction vessel contained an identical solution except that it
lacked .beta.-NAD.sup.+. Forty .mu.L of the initial reaction
mixture were added to each of the second and third vessels, which
were then incubated at room temperature for one hour. The
absorbance of both the second and third reactions was recorded at
340 nM. The third reaction was treated as a baseline, and its
absorbance was subtracted from that of the second reaction prior to
further data analysis.
[0199] All Other Compounds
[0200] Small molecule inhibition and binding titrations with
compounds SU-1, SU-2, SU-4, SU-5, SU-7, SU-8, and SU-10-SU-36 (see
FIG. 9 for structures), compound 968, compound 031, and compound 27
were performed in a 96-well format in a Tecan Saphire absorbance
and fluorescence plate reader. The activity of GAC was measured in
a coupled assay, by monitoring the NADH produced by glutamate
dehydrogenase, which converts the product of the
glutaminase-catalyzed reaction, glutamate, to .alpha.-ketoglutarate
and ammonia by reducing NAD+ to NADH. Because solutions containing
glutamine undergo non-enzymatic degradation to glutamate, samples
were further analyzed by subtracting the NADH produced by
glutaminase in the presence of 968, BPTES, or the equivalent volume
of DMSO as a control, from the NADH produced in the absence of
glutaminase under identical experimental conditions. NADH was
quantified using a standard curve of freshly prepared NADH (Sigma)
in 50 mM Tris-Acetate, pH 8.5, and 0.1 mM EDTA.
[0201] Detailed procedures for the real-time binding and inhibition
assays adapted for 96-well microtiter format are as follows: 2
.mu.L of inhibitor or DMSO was distributed across the 96-well
plate, followed by addition of 200 .mu.L 10 nM 488-GAC, unlabeled
WT-GAC, or no added GAC as a negative control, in 50 mM
Tris-Acetate, pH 8.5, and 0.1 mM EDTA, followed by immediate
monitoring of 488 fluorescence (490 nm/520 nm excitation/emission,
5 nm/20 nm excitation/emission slits). The 488-fluorescence was
measured every 2 minutes with 90 seconds of orbital shaking,
followed by 30 seconds resting between each cycle for a total of
four cycles (i.e. 6 minutes). A mixture of GDH and NAD+(20 .mu.L)
was then added to give 10 units of GDH and 2 mM NAD+. To activate
GAC, 30 .mu.L of a mixture of glutamine and K.sub.2HPO.sub.4 was
added to give a total concentration of 50 mM K.sub.2HPO.sub.4 and
20 mM glutamine in each well. NADH fluorescence was measured (340
nm/460 nm excitation/emission, 10 nm/10 nm excitation/emission
slits) every minute with 30 seconds orbital shaking, and a 30
second rest between each reading, for 10 cycles (i.e. 9 minutes).
Three wells were prepared for each experimental condition (i.e.
each concentration of compound) alongside one well where 2 .mu.L of
DMSO was added in place of inhibitor, and one well that contained
the small molecule inhibitor but no GAC. To analyze 488-quenching
by the added compound, 488-fluorescence (F) was normalized to the
DMSO control (F0). Quenching was quantified as follows: 1-F/F0. For
compounds that emitted fluorescence within the observed range,
fluorescence measured in the well that contained the compound but
lacked GAC was used to subtract added fluorescence due to the
compound. Similarly, samples were analyzed for NADH fluorescence by
subtracting the fluorescence measured for the experimental
condition from the NADH fluorescence in the well that contained the
added compound but no GAC. Percent inhibition at each drug
concentration was calculated using the adjacent DMSO control. FIGS.
10A-10AJ show both quenching and inhibition results for various
inhibitors of GAC in vitro. The compounds were ranked in order of
their potency as shown in FIG. 11. The correlation between
IC.sub.50 and K.sub.D of the GLS1 inhibitors is shown in FIG.
12.
[0202] Certain of the inhibitors were also tested for their ability
to inhibit the KGA splice variant, using the same method described
supra but using 488-KGA in place of 488-GAC. FIGS. 13A-F show both
quenching and inhibition results for various inhibitors of KGA in
vitro.
Example 18--Cell-Based Assay
[0203] MDA-MB-231 cells were cultured in RPMI-1640 media
supplemented with 10% FBS. Prior to initiating the assay, cells
that were 70-80% confluent were trypsinized, diluted, counted, and
dispensed into 6-well culture plates at a density of
1.times.10.sup.4 cells per well. Each well was then brought to 2 mL
of media, total. The cells were allowed to adhere to the wells
overnight. At this time, and every 72 hours thereafter, media was
exchanged for media containing either the indicated amount of a
given inhibitor, diluted from an appropriate DMSO stock, or an
equivalent amount of DMSO without inhibitor. Cells were counted on
the 6th day of culture. Cell counting was performed by aspirating
media, rinsing the cells with room temperature PBS, and then
incubating at 37.degree. C. for 5 minutes in 0.5 mL trypsin-EDTA
solution. The culture plates were then agitated to fully dissociate
cells from the plate surfaces, and 0.5 mL of RPMI-1640 complete
media were added to quench trypsin activity. Cells were then
counted on a hemocytometer, with 3 readings taken and averaged per
sample. All experiments were performed in triplicate. Dose curves
and IC.sub.50 values were determined in Sigmaplot. IC.sub.50 values
are shown in FIG. 14. Using this assay, inhibitory properties of
SU-16 to SU-20 were evaluated and are shown in FIG. 15.
Example 19--Covalent Cross Linking of Photo Reactive Compounds in
Vitro and in Cells
[0204] Glutaminase proteins of the WT sequence, or containing the
mutations D391K alone, or the mutations K316E/D391K/R459E, were
purified as detailed in Example 3 supra. These glutaminase proteins
were incubated with increasing concentrations of the compound
SU-22, and excited with a hand held UV lamp for 120 seconds.
Immediately following UV exposure, samples were loaded onto a 4-20%
Tris-Glycine polyacrylamide gel after the addition of SDS-PAGE
sample buffer (50 mM Tris-HCl (pH=6.8), 2% SDS, 10% glycerol, 1%
.beta.-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue), and
visualized on a UV light box.
[0205] Conditions of in vitro cross linking of WT GAC, and the
K316E/D391K/R459E GAC mutant were adapted for efficient cross
linking and purification of the cross linked protein product. WT
GAC and the K316E/D391K/R459E GAC mutant, both containing the
N-terminal His-tag, were diluted to a concentration of 100 nM in 20
mM Tris-HCl (pH=8.6) 100 mM NaCl, upon which 10 .mu.M of SU-22 was
added. Following a preincubation time of 5 minutes, samples were
exposed to UV light under a hand held 500 W UV lamp to initiate the
photo cross linking. Immediately following, bovine serum albumin
(BSA, Sigma) was added to a final concentration of 2 mg/mL to help
stabilize the cross linked product and to bind excess un-reacted
SU-22. To isolate the cross linked product, 2.5 mg cobalt agarose
beads (Clontech) were added and the solution was gravity filtered.
The beads were first washed with 10 column volumes of 50 mM
Tris-HCl (pH=8.6) 10 mM NaCl, 5 column volumes 50 mM Tris-HCl
(pH=8.6) 10 mM NaCl containing 5 mM imidazole, 3 column volumes 50
mM Tris-HCl (pH=8.6) 10 mM NaCl containing 20 mM imidazole, and
finally the cross linked product was eluted using 4 column volumes
of 50 mM Tris-HCl (pH=8.6) 10 mM NaCl containing 320 mM imidazole.
The purified cross linked protein was then concentrated using spin
columns with a 30 kD molecular weight cutoff and quantified using
UV-vis spectroscopy, where the protein was quantified using the
A280 (.epsilon.280=38,850 M-1 cm-1) and the cross-linked SU-22
using the A350 (.epsilon.350=18,900 M-1 cm-1). Ratios of cross
linked SU-22 to WT and K316E/D391K/R459E GAC were 0.52 and 0.78,
respectively.
[0206] To further illustrate specific cross linking, the cross
linked K316E/D391K/R459E was analyzed using analytical size
exclusion chromatography, where 100 .mu.g of cross linked
K316E/D391K/R459E was injected onto a superdex 200 10/300 GL (GE
Healthcare) equilibrated in 50 mM Tris-HCl (pH=8.6) 20 mM NaCl with
a flow rate of 0.3 mL/min monitoring both the absorbance
wavelengths at 280 nm and 350 nm.
[0207] The cross linked WT GAC was subjected to an overnight
typsinization by first reacting all purified cross linked WT GAC
(2.5 mg) with 10 mM iodoacetamide, followed by addition of 25 .mu.g
sequencing grade trypsin (Roche) and rotated overnight at
37.degree. C. Following overnight trypsin digestion the solution
was filtered using a 10 kD MW cutoff spin filter and injected onto
a SunFire C18 100 .ANG. 5 .mu.m, 4.6 mm.times.150 mm (Waters).
Peptides were eluted using a binary gradient elution protocol,
where mobile phase A (5:95:0.1 acetonitrile:water:trifluoroacetic
acid) and mobile phase B (95:5:0.1
acetonitrile:water:trifluoroacetic acid) were varied to produce an
increase in acetonitrile up to 80% over 20 minutes at a flow rate
of 1 mL/min. The wavelengths 254 nm and 35 nm were monitored.
[0208] Cross linking of SU-22 was also performed in cells
transformed by the oncogene, Dbl. For these experiments,
Dbl-induced MEFs, as detailed in Example 1 supra, were cultured in
150 mm.sup.2 dishes to approximately 80% confluency in DMEM
supplemented with 10% FBS. Cells were then washed and DMEM
supplemented with 1% FBS and 5 .mu.M SU-22 was added, and cells
were cultured overnight. Cells were then exposed to 60 seconds of
UV light by a hand held 50 W UV lamp to initiate the in cell cross
linking, followed by washing with DMEM supplemented with 1% FBS
without SU-22. Cells were incubated with DMEM supplemented with 1%
FBS following UV exposure for 20 minutes. The cells were then
trypsinized, and mitochondria were isolated as outlined in Frezza
et al., "Organelle Isolation: Functional Mitochondria from Mouse
Liver, Muscle, and Cultured Fibroblasts" Nature Protocols 2:287-95
(2007), which is hereby incorporated by reference in its entirety.
Briefly, suspension of trypsinized cells were spun 600.times.g 10
minutes at 4.degree. C. and suspended in mitochondrial isolation
buffer (10 mM Tris-MOPS (pH=7.4), 1 mM EGTA/Tris, and 200 mM
sucrose). Cells were homogenized in a glass potter with a Teflon
pestle using a Dounce homogenizer operated at approximately 1600
rpm for 35 strokes. The homogenate was centrifuged 600.times.g 10
minutes at 4.degree. C. and the supernatant was isolated. The
pellet was lysed in RIPA lysis buffer and used as the P1 fraction
containing the nucleus and unbroken cells. The supernatant was spun
at 7000.times.g 10 minutes at 4.degree. C., where the resultant
pellet was the isolated mitochondria and the supernatant comprising
the soluble cytosolic and microsomal fractions. The supernatant was
isolated and spun at 200,000.times.g 1 hour at 4.degree. C., and
the resulting pellet was taken to be the 5200 fraction containing
cytosolic and microsomal components. The mitochondrial pellet was
resuspended in mitochondrial isolation buffer and spun again at
7000.times.g 10 minutes at 4.degree. C., where the resulting pellet
was taken to be the purified isolated mitochondria.
[0209] For experiments where cells were visualized using confocal
fluorescence microscopy, cells were cultured in the same conditions
as stated above in MakTek 50 mm.sup.2 dishes. Cells were treated
with 5 mM SU-22 overnight in DMEM supplemented with 1% FBS.
Following 30 seconds of UV exposure, media was replaced with fresh
DMEM (1% FBS) and incubated at 37.degree. C. for 2 hours. The media
was then switched to DMEM (1 FBS) containing 250 nM of the
mitochondrial fluorescent probe MitoTracker CMXRos and incubated at
37.degree. C. for 30 minutes, at which point the media was changed
to DMEM (1% FBS) without the MitoTracker for an additional 30
minutes. Cells were then fixed in 4% para-formaldehyde and 0.1%
glutaraldehyde and imaged on an inverted Axio Observer.Z1
microscope using the 405 laser line for excitation of the SU-22
small molecule and 514 laser line to excite the MitoTracker.
[0210] For experiments using the novel compound SU-34, which
contains both a photo-cross linking moiety along with a click
chemistry ready alkyne functionality, cells were treated the same
as stated above. Dbl-transformed cells were seeded in 150 mm.sup.2
dishes and cultured overnight in DMEM supplemented with 1% FBS and
5 mM SU-34. Following 60 seconds UV exposure cells were harvested
and mitochondria were isolated as described above. To each
subcellular fraction, the click chemistry was performed by adding
reagents to give a final concentration as follows: 20 mg protein,
100 mM MOPS (pH=7), 0.1 mM CuSO.sub.4 and 0.5 mM
Tris(3-hydroxypropyltriazolyl-methyl)amine (THPA) premixed, 5 mM
ascorbate, 20 mM Alexa488-azide (molecular probes). The reaction
was performed at 37.degree. C. for 1 hour on a rotisserie inverter.
SDS-PAGE sample buffer was added and each sample was run on a 4-20%
Tris-glycine SDS-PAGE. The gel was first imaged for in gel
fluorescence using the GelDoc XR+ molecular imager (Biorad),
followed by transfer to PVDF and immunoblotting with the GLS1
antibody (AP8809b, Abgent).
[0211] FIGS. 16A-B show both SU-22 quenching and inhibition results
following UV exposure.
Discussion of Examples 16-19
[0212] In vitro inhibition and binding assays of newly synthesized
kidney-type glutaminase inhibitors are illustrated in FIGS. 9
through 13. These were obtained using the methods described in
Stalnecker et al., "Mechanism by Which a Recently Discovered
Allosteric Inhibitor Blocks Glutamine Metabolism in Transformed
Cells," PNAS 112(2):394-99 (2013), which is hereby incorporated by
reference in its entirety, where a fluorescently labeled GAC or KGA
isoform was assayed first for the binding of these novel inhibitors
followed by assaying the activity of the enzyme. FIG. 9 represents
the in vitro inhibition of 968-like compounds as well as the novel
scaffold described in this invention. FIG. 11 represents the in
vitro inhibition data from FIG. 9 in a histogram, where trends are
more clearly illustrated. Compounds SU-24, SU-15, SU-22, SU-1,
SU-14, SU-10, SU-29, SU-20, and SU-31 inhibited the GAC isoform of
the kidney-type glutaminase at a lower concentration than the
parent compound, 968. Of these compounds, SU-29, SU-20, and SU-31
were derivatives of the novel scaffold disclosed in this invention,
and represent model compounds for effective non-968 inhibitors of
kidney-type glutaminase. The binding and inhibition data for these
inhibitors were plotted on a two dimensional graph in FIG. 12,
where a direct correlation of the binding of these small molecules
(y-axis) and inhibition (x-axis) is illustrated. These results
provide evidence for the novel compounds disclosed in this
invention to bind to the same proposed binding site for the
original small molecule 968, and demonstrate the resultant
inhibition of enzymatic activity upon binding. The complete data
set for each compound analyzed in this in vitro binding and
inhibition assay for both 488-labeled GAC and 488-labeled KGA
isoforms are presented in FIGS. 10A-AJ (GAC binding/inhibition),
FIGS. 13A-F (KGA binding/inhibition), and FIGS. 16A-B (SU-22
quenching/inhibition following UV exposure). FIGS. 14 and 15
demonstrate the ability of the compounds disclosed in this
invention to inhibit the growth of cancer cells. Thus, the
compounds that are able to inhibit the enzyme glutaminase in vitro,
are able to inhibit cancer cell growth by inhibiting the
glutaminase enzyme present in cancer cells.
[0213] The use of the novel photo-cross linking 968 derivatives, as
disclosed here, was investigated both in vitro and in cells. FIG.
17 illustrates the cross linking of SU-22 with the WT GAC isoform
of GLS1, along with the mutants D391K, previously shown to be a
constitutive dimer using the methods described above in Example 7,
and K316E/D391K/R459E, shown to be a constitutive monomer.
Consistent with previously published results (Stalnecker et al.,
"Mechanism by Which a Recently Discovered Allosteric Inhibitor
Blocks Glutamine Metabolism in Transformed Cells," PNAS
112(2):394-99 (2013), which is hereby incorporated by reference in
its entirety), the cross linking of the SU-22 was shown to be more
efficient for the monomer mutant, i.e. K316E/D391K/R459E, when
compared to the dimer (D391K), or WT GAC. The dose dependent cross
linking was consistent with the inhibition profile of SU-34 for WT
GAC, included here in Figure LOAF. The methods for cross linking
SU-22 to GAC in vitro were extended to develop a purification
protocol of cross linking SU-22 to GLS1 proteins (see FIGS. 18A-D).
The purification utilized the N-terminal 6.times.-His tag on
purified GLS1 constructs, where the protein of interest was
incubated with SU-22 and cross linked using UV irradiation. The
protein SU-22 conjugate was then purified using cobalt agarose
beads, where the elution of the labeled GLS1 protein was monitored
by running each fraction on an SDS-PAGE and exciting with UV light
to visualize the fluorescent SU-22 molecule. FIG. 18A illustrates
the fluorescence of each fraction of the purification protocol for
cross linking SU-22 to the K316E/D391K/R459E GAC mutant, where
strong fluorescence was visualized in the elution fractions, but
not the original sample (compare lanes 1 and 8). Additionally, the
total protein in each fraction was visualized using Coomassie blue
staining, further illustrating the purification of the SU-22
labeled species. The isolated SU-22 conjugate was further analyzed
using analytical gel filtration, as illustrated in FIG. 18B, where
the absorbance trace at 280 nm represents the elution of the
protein species and the absorbance trace at 350 nm represents the
absorbance of the small molecule, SU-22. Two distinct peaks are
observed, corresponding to the monomer and dimer of GAC, consistent
with this small molecule binding at the interface between two
monomers as shown previously. This method was extended to include
the WT GAC protein, where the final protein SU-22 conjugate was
analyzed using UV-vis spectroscopy. FIG. 18C illustrates the
absorbance profile of the isolated SU-22 WT GAC conjugate, where
the absorbance at 350 nm is characteristic of the small molecule
SU-22. Additionally, this SU-22 WT GAC conjugate was subjected to
further analysis and characterization by trypsin digestion and
subsequent analysis of the resultant peptides by HPLC. FIG. 18D
illustrates the HPLC profile of the WT SU-22 conjugate, where the
absorbance trace at 254 nm represents any eluted peptides, and the
absorbance at 350 nm represents the small molecule SU-22 conjugated
peptide (arrow). These results represent general methods for the
covalent modification and purification of GLS1 proteins with the
photo-cross linker described here, SU-22.
[0214] Experiments were conducted to also analyze these novel GLS1
inhibitor photo-cross linkers in cells. FIG. 19 illustrates
confocal microscopy images of fixed Dbl-transformed MEFs following
UV stimulation, which initiates the cross linking of SU-22 in
cells, and subsequent washing of excess unreacted SU-22 followed by
labeling of cellular mitochondria with the reliable MitoTracker
probe. These images demonstrate the subcellular localization of the
SU-22 small molecule, where co-localization is observed to be most
consistent with mitochondrial labeling. Additionally, these methods
of cross linking SU-22 in cells was extended to purify conjugated
SU-22 proteins. FIG. 20 illustrates the subcellular fraction of
Dbl-transformed cells cross linked with SU-22, where the each
fraction was analyzed by SDS-PAGE. SU-22 conjugated proteins were
visualized by illumination of the gel by UV light, where distinct
bands could be visualized. The gel was then transferred to PVDF
membrane and analyzed by immunblotting with the GLS1 antibody, and
similar banding patterns were found to be characteristic of SU-22
cross linked GLS1 proteins (white box). These results also further
suggest the localization of this small molecule to the
mitochondrial fraction.
[0215] In order to investigate the use of these novel GLS1
inhibitor cross linkers, these methods were extended for the use of
the novel SU-34 cross linker. This cross linker contains both a
photo reactive moiety and a click chemistry ready alkyne functional
group. FIG. 21 illustrates a cross linking experiment, where
Dbl-transformed cells were incubated with the SU-34 compound and
cross linked following UV exposure. Following the cross linking,
the cells were subfractionated as previously described and samples
from each fraction were reacted using click chemistry to the highly
fluorescent Alexa488 azide probe. The fractions were then analyzed
by SDS-PAGE and SU-34 cross linked proteins, following the addition
of the highly fluorescent Alexa488 azide via click chemistry, were
visualized by fluorescence under UV excitation (white box). These
samples were then transferred to PVDF membrane and immunoblotted
using a GLS1 antibody. These results are consistent with those
included for the SU-22 photo-cross linker, where the fluorescence
of the cross linked small molecule protein conjugate is consistent
with the labeling of GLS1 proteins in cells.
[0216] In conclusion, binding and inhibition data for the new
compounds illustrated in FIG. 9 were obtained using the
fluorescently labeled GAC and KGA isoforms. The data presented in
Examples 16-19 demonstrate the efficacy of these novel compounds,
and present evidence for these inhibitors to occupy the same
binding site as the parent compound, 968. Indeed, a novel inhibitor
scaffold is described, where the efficacy of such was shown to be
as potent, or in some derivatives more potent, than 968 itself.
Additionally, a class of 968-derivatives that contain functional
groups that allow for the covalent attachment of the small molecule
to the protein target are described. These compounds were shown to
inhibit GLS1 in the same manner as the parent compound, 968.
Furthermore, the covalent cross linking and purification of protein
conjugates both in vitro and in vivo are described, along with
methods of analysis to assess the function or subcellular
localization. Combined, these novel compounds represent a new class
of inhibitors of GAC activity.
* * * * *